Synthesis and cytotoxic activity of a small naphthoquinone library: First synthesis of juglonbutin

Article abstract of DOI:10.1002/ejoc.201402272

A synthetic protocol has been designed to synthesize grecoketidone (2k), 5-hydroxylapachol (2g), and the recently discovered natural products juglonbutin (2o) and its derivatives, leading to a small library of different 1,4-naphthoquinones with the intention of finding new active compounds. Within our collection, 2-O-alkylated naphthoquinones with an ester functionality in the side-chain and a free OH group at C-5 showed the best activities. Compounds 2f, 2m, and 2n showed GI₅₀ values against 12 tumor cell lines in the lower micromolar range and juglonbutin (2o) showed remarkably efficient inhibition of the glycogen synthase kinase 3β with an IC₅₀ value of 2.03 μM. Furthermore, studies on the mode of action of the most active cytotoxic compounds have been carried out. To the best of our knowledge, this is the first report on the synthesis of juglonbutin (2o) and its biological activity. Copyright

Full text of DOI:10.1002/ejoc.201402272

FULL PAPER
DOI: 10.1002/ejoc.201402272
Synthesis and Cytotoxic Activity of a Small Naphthoquinone Library: First
Synthesis of Juglonbutin
Elke Brötz,[a][‡] Jennifer Herrmann, Jutta Wiese, Heidi Zinecker,
[b]
[c]
[c][‡‡]
Armin Maier,
[d]
[d]
[c]
[b]
[a]
Gerhardt Kelter, Johannes F. Imhoff, Rolf Müller, and Thomas Paululat*
Dedicated to Professor Axel Zeeck on the occasion of his 75th birthday
Keywords: Natural products / Quinones / Medicinal chemistry / Toxicity / Antitumor agents
A synthetic protocol has been designed to synthesize greco-
ketidone (2k), 5-hydroxylapachol (2g), and the recently dis-
covered natural products juglonbutin (2o) and its derivatives,
leading to a small library of different 1,4-naphthoquinones
with the intention of finding new active compounds. Within
our collection, 2-O-alkylated naphthoquinones with an ester
functionality in the side-chain and a free OH group at C-5
showed the best activities. Compounds 2f, 2m, and 2n
showed GI50 values against 12 tumor cell lines in the lower
micromolar range and juglonbutin (2o) showed remarkably
efficient inhibition of the glycogen synthase kinase 3β with
an IC50 value of 2.03 μM. Furthermore, studies on the mode
of action of the most active cytotoxic compounds have been
carried out. To the best of our knowledge, this is the first
report on the synthesis of juglonbutin (2o) and its biological
activity.
Introduction
Other naphthoquinones show cytotoxic activities, for ex-
ample, the rhinacanthins show activity against different
tumor cell lines. Lapachol is described as a potential anti-
Screening for new anticancer drugs is still important and
is one of the major goals in medicinal chemistry.[1] Second-
ary metabolites from the naphthoquinone family and deriv-
atives thereof show different (antibacterial, cytotoxic) bio-
logical activities or interact with different enzymes.[ Exam-
ples of antibacterial naphthoquinones are javanicine, which
is active against different Pseudomonas species, or juglomy-
cin Z, which shows activity against Gram-positive and
[6]
cancer agent and derivatives of lapachol show a wide range
[1,7]
of different biological activities.
5-Hydroxylapachol, iso-
lated from the root heart wood of Tectona grandis, shows
2]
[8]
cytotoxic activity towards Artemia salina (brine shrimp).
The synthesis and antiproliferative activity of 5-hydrox-
ylapachol and related naphthoquinones have been reported,
and the 5-hydroxy group is described as being very impor-
[3,4]
Gram-negative bacteria.
Antifungal activity is described
[9,10]
tant for the antiproliferative effect.
Moreover, the
for hydroxysesamone.[
5]
highly cytotoxic activity of 5-methoxyjuglone is reported to
be caused by the induction of apoptosis due to the acti-
[
11,12]
[
a] Institut für Organische Chemie II, Universität Siegen,
Adolf-Reichwein-Str. 2, 57076 Siegen, Germany
E-mail: thomas.paululat@uni-siegen.de
vation of different caspases.
We became interested in the synthesis of naphtho-
quinones and their screening for biological activity after
finding the new natural products grecoketides A and B pro-
http://www.chemie-biologie.uni-siegen.de/oc/oc2/mitarbeiter/
paululat/
[
b] Helmholtz Institut für Pharmazeutische Forschung Saarland,
Helmholtz Zentrum für Infektionsforschung und Institut für
Pharmazeutische Biotechnologie, Universität des Saarlandes,
Postfach 151150, 66041 Saarbrücken, Germany
c] GEOMAR Helmholtz-Zentrum für Ozeanforschung Kiel,
Universität Kiel,
[
13]
duced from Streptomyces sp. Acta 1362.
Although no
biological activity was found for either grecoketide, the
aglycon grecoketidone (2k) is strongly related to the known
active natural products 5-hydroxylapachol (2g), gonio-
[
Düsterbrooker Weg 20, 24105 Kiel, Germany
d] Oncotest GmbH,
Am Flughafen 12–14, 79108 Freiburg, Germany
‡] Present adress: Helmholtz Institut für Pharmazeutische
Forschung Saarland,
[8,14,15]
quinone, and other naphthoquinones.
[
[
Thus, one of our interests was to evaluate the activity
profile of grecoketidone (2k) in comparison with 5-hydrox-
ylapachol (2g). Therefore we planned to synthesize these
Postfach 151150, 66041 Saarbrücken, Germany
[
‡‡] Present address: Pharmaceutical Institute, Pharmaceutical two naphthoquinones. Both the naphthoquinone products
Chemistry I,
and the side-products arising from the synthesis form a
small library of compounds that have been screened for
their biological activity in different test systems. As the re-
An der Immenburg 4, 53121 Bonn, Germany
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201402272.
318
5
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 5318–5330

First Synthesis of Juglonbutin
cently published juglonbutin (2o) should be a realistic target chlorinated byproduct 2p after addition of γ-aminobutyric
for synthesis by our approach, we became interested in its acid to 2-chlorojuglone (2b; Scheme 2, h). The 5-O-alk-
synthesis for activity screening.[ The first results of the oxylated methoxyjuglone derivative 2q was synthesized
screening led us to focus on the cytotoxicity of naphtho- from 2-methoxyjuglone (2c) by reaction with ethyl bromo-
16]
quinones.
acetate (Scheme 2, j).
We used naphthazarin (4a) as the starting material for
the synthesis of compound 4e, with two ester-functionalized
chains at C-5-O and C-8-O (Scheme 3). Methylation of the
two hydroxy groups gave 5,8-dimethoxynaphthazarin (4b).
The methoxy group was introduced at C-2 of 4b by using
Results and Discussion
Chemistry
[20]
a suspension of dioxane/methanol with H SO /Fe SO .
2
4
2
4
We synthesized 2-hydroxyjuglone (2d) as the key interme-
Deprotection of 2,5,8-trimethoxy-1,4-naphthoquinone (4c)
diate for the introduction of the desired side-chain at C-3
at C-5-O and C-8-O with HNO and AgO gave compound
in five steps (Scheme 1).[17] The synthesis started from 1,5-
3
4d, which was treated with ethyl iodoacetate to give the tar-
dihydroxynaphthalene (1a). Acetylation of the hydroxy
groups in acetic acid anhydride led to 1,5-diacetoxynaphth-
alene (1b), which was oxidized and chlorinated by N-
chlorosuccinimide to give 5-acetoxy-2-chloro-1,4-naphtho-
quinone (2a). Naphthoquinone 2a was deprotected with
get compound 4e.
In contrast to the good yields obtained in the synthesis
of the key intermediate 2d, the introduction of side-chains
was only achieved in low yields, in agreement with the find-
[
8,19,21,22]
ings of other groups.
One possible reason for this
AlCl to yield 2-chlorojuglone (2b). Treatment of 2-chloro-
3
behavior is the mesomeric stabilization of the deprotonated
juglone (2b) in alkaline methanol solution provided 2-meth-
oxyjuglone (2c), which was converted by hydrolysis into 2-
hydroxyjuglone (2d).
naphthoquinone nucleophile of 2d, which may inhibit selec-
[
23]
tive substitution.
The phenolic hydroxy group at C-5 is
presumably chelated, which may protect that position
towards substitution. Only a huge excess of bromoalkane
prevents the formation of much more complex reaction
mixtures, as in the reaction using ethyl bromoacetate lead-
ing to 2j. Compound 2p is presumably the result of a
Michael addition reaction, followed by reduction to the hy-
droquinone and subsequent oxidation to the quinone.
Scheme 1. Synthesis of the key intermediate 2-hydroxyjuglone (2d).
Reagents and conditions: (a) Ac
b) N-chlorosuccinimide, CH COOH/H
Cl , r.t., 3 h, 2. Ice/H
OH, r.t., 2 h, 85%; (e) K
2
O, pyridine, 35 °C, 1 h, 66%;
O, 50 °C, 1 h, 70–75 °C,
O, CH Cl , r.t.,
CO , CH OH/
(
1
3
3
2
Antibacterial Activity and Biochemical Tests
.5 h, 53%; (c) 1. AlCl
h, 96%; (d) NaOH, CH
O, 80 °C, 40 min, 92%.
3
, CH
3
2
2
2
2
2
2
3
3
First-order profiling of our library was conducted in dif-
ferent antibacterial agar diffusion assays as well as target-
H
2
The alkylation of 2b–2d by different substitution reac- based and cell line assays (detailed results are given in the
tions gave naphthoquinone derivatives in one step, with the Supporting Information). The tested naphthoquinones
exception of 2k, synthesized from 2d, which required two showed good antibacterial activity against the Gram-posi-
steps (Scheme 2). For the alkylation at the C-3 position to tive bacteria B. subtilis and S. lentus, but most showed only
prepare 2g and 2k, we used a synthetic procedure that had moderate activities in all other tests. Higher activities were
[
18]
been used for lapachol but with a slight modification.
observed for naphthoquinones with chlorine or O-alkyl-
Under these conditions we obtained 2g, together with the ation at the C-2 position. The 2-O-ester-functionalized
two byproducts 2h and 2i, similar to the experience of Boni- naphthoquinone 2m showed pronounced activity against
fazi et al. (Scheme 2, b).[ The precursor molecule of greco- Staphylococcus epidermidis DSM 3269 with an IC50 value
ketidone (2k), grecoketidone ethyl ester (2e), was obtained of 0.51 μm. It should be highlighted that four compounds,
under these conditions together with 2f in low yields 2c and 2f–2h, showed similar MIC values against Staphylo-
8]
(
Scheme 2, a). More basic reaction conditions led to the coccus aureus MRS3 in the range of 0.5–2.0 μg/mL, like the
[19]
formation of 2e together with 2j (Scheme 2, c).
Similar reference antibiotics chloramphenicol, kanamycine, and
conditions led to the synthesis of 2l (Scheme 2, e). The reac- gentamycine. Juglonbutin (2o) showed inhibition of glyco-
tion of 2-chlorojuglone (2b) with ethyl glycolate led to 2f as gen synthase kinase 3β (GSK3β) with an IC50 value of
a single product (Scheme 2, i). The related 1,4-naphtho- 2.03 μm.
quinones 2m and 2n were synthesized starting from 2-
GSK3 is a therapeutic target for the treatment of diabe-
hydroxyjuglone (2d) by using the corresponding iodo ester tes, inflammation, and multiple neurological diseases.[
as alkylating reagent because hydroxylated ethyl propanoate Compounds 2j and 4e showed inhibitory effects on the pro-
and butanoate were not accessible (Scheme 2, f,g). The syn- tein tyrosine phosphatase 1B (55% inhibition with c =
thesis of 2n also gave 1,2-naphthoquinone 3a as a byprod- 10 μm), which is a promising target for the treatment of
24]
[
25,26]
uct. Juglonbutin (2o) was synthesized together with the type 2 diabetes, obesity, and cancer.
Eur. J. Org. Chem. 2014, 5318–5330
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5319

T. Paululat et al.
FULL PAPER
2 2 5
Scheme 2. Synthesis of naphthoquinones 2e–2q. Reagents and conditions: (a) LiH, DMSO, –78 °C, r.t., KI, BrCH COOC H , 55 °C, 4 h,
9
(
% (2e), 26% (2f); (b) 1. LiH, DMSO, –78 °C; 2. KI, BrCH
c) K CO
propanone, DMF, r.t., 3 d, 27% (2l); (f) Li
CHCl , reflux, 2 d, 8% (2n), 4% (3a); (h) K
ethyl glycolate, CH CN, 70 °C, 25 h, 30% (2f); (j) K
2
CH=C(CH
3
)
2
, r.t.; 3. 45 °C, 4 h, 4. 15 h r.t., 28% (2g), 2% (2h), 8% (2i);
CO , bromo-2-
O, ethyl 3-iodopropanoate,
O, reflux, 8 h, 19% (2o), 27% (2p); (i) 2 n NaOH,
3
, ethyl bromoacetate, DMF, r.t., 20 h, 38% (2q).
2
3
, ethyl bromoacetate, DMF, r.t., 3 d, 3% (2e), 9% (2j); (d) 2 n NaOH, EtOH, r.t., 40 min, 89% (2k); (e) Li
CO , ethyl 4-iodobutanoate, DMF, r.t., 17 d, 44% (2m); (g) Ag
CO , γ-aminobutyric acid, CH CN/H
CO
2
3
2
3
2
3
2
3
3
2
3
2
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Eur. J. Org. Chem. 2014, 5318–5330

First Synthesis of Juglonbutin
Table 1. Antitumor potency of 2e–q, 3a, and 4e against 12 cell lines based on GI50 values.
GI50 [μm]
Cell line: HT29
251L
529L
629L
401NL
462NL
889L
1657L
22RV1
1752L
486L
1138L
2
2
2
2
2
2
2
2
2
2
2
2
3
4
e
Ͼ30
10.1
26.9
Ͼ30
Ͼ30
Ͼ30
Ͼ30
2.13
1.22
28.9
13.2
9.37
10.2
3.42
Ͼ30
8.11
38.7
35.5
17.9
9.37
Ͼ30
2.87
2.43
26.0
9.89
14.4
12.0
6.85
Ͼ30
1.02
14.6
5.99
26.4
7.49
Ͼ30
2.24
1.17
25.8
10.1
6.77
3.11
3.62
Ͼ30
0.96
8.16
9.49
28.6
9.02
51.7
1.62
1.34
17.7
7.55
5.02
7.65
5.14
Ͼ30
1.70
26.6
22.4
33.0
8.77
Ͼ30
3.53
2.79
22.7
10.7
10.9
7.34
5.25
Ͼ30
8.14
1.66
12.9
15.2
8.67
Ͼ30
5.26
4.10
26.8
7.71
13.0
11.7
4.93
Ͼ30
6.87
Ͼ30
42.9
51.2
10.37
Ͼ30
3.45
2.15
29.6
26.4
23.3
8.20
8.32
Ͼ30
7.54
67.2
10.9
52.6
9.72
Ͼ30
2.59
1.79
29.6
10.5
7.69
3.63
3.58
Ͼ30
3.58
9.49
20.6
17.8
6.33
Ͼ30
3.25
2.68
29.5
9.10
8.32
8.86
6.70
Ͼ30
4.19
16.0
17.5
25.3
9.49
Ͼ30
2.15
1.43
27.8
12.0
10.1
6.49
2.99
Ͼ30
1.09
23.8
60.9
37.8
9.37
Ͼ30
5.05
3.83
30.0
13.3
14.1
11.5
8.16
Ͼ30
1.13
3.33
8.24
12.6
1.67
Ͼ30
3.08
2.04
29.0
11.0
6.31
4.30
3.33
f
g
h
i
j
k
m
n
o
p
q
a
e
the 2-O-alkylated naphthoquinones bearing an ester func-
tionality inside the chain, those with a free OH group at
C-5 showed higher activities. This supports the proposal
of other research groups that a free OH group at C-5 is
important for the activity of substituted naphtho-
[
8,10,27,28]
quinones.
Comparison between 2q and 4e seems to
suggest that further glycolate groups increase the activity.
In further investigations, the growth inhibitory potentials
of all the compounds were evaluated against human osteo-
Scheme 3. Synthesis of naphthoquinone 4e. Reagents and condi-
tions: (a) CH I, Ag O, CHCl , 60–65 °C, 4 d, 69%; (b) Fe (SO
SO , MeOH, dioxane, reflux, 2 h, 46%; (c) AgO, HNO
one, 0 °C, r.t., 15 min, 27%; (d) ethyl iodoacetate, K CO , DMF,
r.t., 3 d, additional K CO , r.t., 2 d, 25%.
3
2
3
2
4 3
) ,
H
2
4
3
, acet- sarcoma cells U-2 OS, which were then used in further stud-
2
3
ies on the mechanism of action (Table 2). The IC50 value
determined for the ester-functionalized 2-O-alkylated
naphthoquinone 2f is in the low micromolar range.
2
3
Growth Inhibitory Activity of 2f–l Against Human Cancer
Cell Lines
Table 2. Ranking of 1,4-naphthoquinones according to their IC50
values against human osteosarcoma U-2 OS cells used in mecha-
nism of action studies.
Fourteen compounds (2e–k, 2m–q, 3a, 4e) were used for
antitumor screening. The primary screening was conducted
in a 12 cell line test panel involving different types of cancer
Grouping [μm]
Naphthoquinone
IC50 [μg/mL][a] IC50 [μm][a]
cells (colon CXF HT29, stomach GXF 251L, lung LXFA A) 0.1–2.0
29L and LXFL 529L, breast MAXF 462NL, melanoma
2f
0.14
0.22
0.33
0.60
0.60
0.69
1.53
1.70
1.20
5.0
8.08
12.0
27.8
31.4
46.0
0.5
1.07
1.32
1.97
2.07
2.39
4.21
4.33
4.64
16.1
31.3
43.6
107.8
165.0
166.5
2
c
6
2
k
MEXF 462NL, ovary OVXF 899L, pancreas PAXF 1657L,
prostate PRXF 22RV1, mesothelioma PXF 1752L, kidney
RXF 486L, and uterus UXF 1138L). Their antitumor po-
tency was determined on the basis of their growth inhibi-
tion values (GI ; Table 1); some of the compounds show
2
m
B) 2.0–20.0
2n
2q
2j
4
e
5
0
2
2
h
p
growth inhibitory effects with GI50 values in the lower
micromolar range.
C) 20.0–50.0
2i
2
o
D) Ͼ100
2g
2
2
d
e
Mechanism of Action
Within our library of compounds, naphthoquinones sub-
[a] The IC50 values represent the average of two independent mea-
stituted at C-3 showed lower activity than C-2-substituted surements and were determined in a MTT assay; 5 d incubation;
sigmoidal curve fitting.
naphthoquinones. Our lead molecule, grecoketidone (2k),
and its isomer with the ethyl acetate residue at C-3 (2e)
showed the lowest activity. But the same substituent at the
To exclude apoptosis induction related to a disruption of
2-O-position (2f) led to much higher activity. The influence the cytoskeletal function, the compound library was tested
of the chain length at C-2 on antitumor activity was investi- in routine high-content screening (HCS) on actin filaments
gated with compounds 2f, 2m, and 2n. The best activity was (data not shown) and microtubules with the highly active
observed with two methylene groups (2n) between the ether compound 2f and one representative less-active class B
bridge and the ester functionality at C-2. In addition to all compound, 2j (Figure 1).
Eur. J. Org. Chem. 2014, 5318–5330
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T. Paululat et al.
FULL PAPER
Based on the results from the cell cycle histogram we
decided to investigate the impact of the compounds on
DNA, especially on DNA double-strand breaks (DSBs),
which could be one reason for the apoptosis after the G2/
M phase. In further investigations, the compounds were ex-
amined after serial dilutions according to their grouping
(Table 2). Group A was tested from 0.16–5.00 μm, group B
were tested at a 10-fold higher concentration, and groups C
and D were tested at concentrations of 50 and 100 μm. The
DSBs were analyzed on an automated microscope. The
nuclei were segmented on the basis of the Hoechst33342
Figure 1. HCS images of microtubules and nuclei of U-2 OS cells fluorescence and the intensity of phospho-histone H2A·X
treated with 2f or 2j. I) Immunofluorescence α-tubulin (secondary
(
secondary antibody coupled to Alexa488) was determined
antibody coupled to Alexa488). II) Hoechst33342-stained nuclei.
A) Control cells. B) Treatment with 14 μm 2j for 1 d. C) Treatment
with 3.6 μm 2f for 1 d. Scale bar: 100 μm.
in these regions of interest (ROIs) with a BD AttoVision
v1.6.2 instrument.
During the analysis of the DSBs, it became apparent that
some samples led to cell death at high doses, according to
their grouping This was observed for the following com-
pounds: Group A: 2c (5 μm); group B: 2j (50 μm), 2q
Although 2f and 2j do not target tubulin or actin, it was
found that hallmarks typical of apoptosis became apparent
upon treatment overnight. Incubating human U-2 OS os-
teosarcoma cells with 2j (Figure 1, B) caused alterations in
cellular morphology compared with the vesicle control
(50 μm), 2n (50, 25, 12.5, and 6.3 μm) and 4e (50 μm). All
group A compounds except 2k showed DSBs (Figure 3).
Group A member 2c showed DSBs at concentrations two
and four times less than 5 μm (Figure 3). Similar behavior
was found for group B members 2j, 2q, and 4e, which also
needed to be diluted to see the DSBs instead of cell death
(Figure 1, A). In detail, some enlarged nuclei were observed
and the number of cells per field of view also decreased.
Treatment with 2f led to an even more pronounced effect
(Figure 1, C). Here, nuclei and cells themselves are clearly
(
(
Figure 1). Group B members 2h and 2p needed high doses
50μm) to introduce DSBs, and group B member 2n showed
enlarged and despite this, some late apoptotic fragmented
nuclei can be observed.
cell death in all cases. Therefore it was decided to test this
compound as a group A compound, but then no DSBs were
observed. All the compounds from groups C and D were
essentially inactive (see the Results in the Supporting Infor-
mation). Finally, the two compounds 2f and 2j, as new com-
pounds, showed in their groups the most pronounced intro-
duction of DSBs observed for phospho-histone H2A·X (see
Figures 3 and 4).
The phenomenon of enlarged nuclei is often ac-
companied by a G2/M cell cycle arrest.[29] Therefore human
U-2 OS osteosarcoma cells were treated for 3 d with com-
pounds 2f and 2j (Figure 2). Indeed, the more active com-
pound 2f showed an accumulation of cells in the G2/M
phase, whereas 2j has no impact on cell cycle progression
in the same concentration range.
Figure 3. Signal of phosphorylated histone determined as the rela-
tive intensity of the secondary Alexa488-coupled antibody in the
nuclear area, which is defined by segmentation on Hoechst33342-
stained nuclei. Values represent the average of mean values of two
independent measurements of selected group A and B compounds.
Hydroxyurea was used as a positive control.
Figure 2. Cell cycle histograms of U-2 OS cells following treatment
with naphthoquinones 2f and 2j. U-2 OS cells were treated for 3 d
To be aware of which mode of action is responsible for
with either 2f, 2j, or 1% (v/v) MeOH (control). After DNA staining the introduction of DSBs, we investigated the influence of
with propidium iodide, cells were analyzed on a Guava EasyCyte
our compounds on topoisomerase II and on their ability to
Plus capillary flow cytometer. In total, 5000 viable cells were mea-
create reactive oxygen species (ROS). Only compound 4e
sured and analysis was conducted with a FlowJo v7.6.1 instrument.
showed the formation of ROS in the cells after treatment
with dichlorodihydrofluorescein diacetate (DCFDA) and
In this way cell debris was excluded and histograms display cell
count against relative fluorescence in the red channel.
5322
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Eur. J. Org. Chem. 2014, 5318–5330

First Synthesis of Juglonbutin
the results of investigations of other groups. Moreover the
5
-hydroxy group is reported to be important for higher
[
8–10,22,27]
cytotoxic activities of naphthoquinones.
The ester
functionality in the side-chain at C-2 could be the impulse
responsible for the higher activity compared with linear
[
33]
chains reported in the literature.
Additional investi-
gations concerning the mode of action showed that in most
cases DNA double-strand breaks are responsible for the
cytotoxicity. The activities of the ester-functionalized 2-O-
alkylated 1,4-naphthoquinones are much higher than the
non-ester-functionalized 2-O-alkylated derivatives. Those
with a free OH group at C-5 are more potent. Additional
ester-functionalized chains on the aromatic ring system in-
Figure 4. Exemplary images of HCS on histone H2A·X phosphor-
ylation. I) Control. II) Treatment for 1 d at 14 μm 2j. III) Treatment
for 1 d at 7 μm 2f. The upper right image displays in detail cells creases the activity. Although the quinones are all structural
treated with 2f showing H2A·X foci. Scale bar: 100 μm.
related, a common mode of action could not be found. For
example, the main related compounds 2f, 2m, and 2j show
the introduction of DSBs, but not 2n. The two highly active
comparison with tert-butyl hydroperoxide (tBHP) as refer-
compounds 2f and 2j are not involved in the production of
ence (Figure 5). Naphthoquinones 2c, 2f, 2g, and 2j showed
ROS, but they show strong inhibitory effects in the topoiso-
higher inhibitory activity on topoisomerase II than the ref-
merase II assay, leading to the conclusion that this is the
erence compound etoposide (see Table S5 in the Supporting
major target of these naphthoquinones.
Information), which led to the conclusion that topoisomer-
ase II is the major target of these naphthoquinones, which
is in good agreement with the inhibition of topoisomer-
ases I and II reported in the literature.[
19,30–32]
Experimental Section
Chemistry
General: Reagents and solvents were commercially available and
used without further purification. Reactions were monitored by
TLC using plates from Merck (KG 60 F254, 0.2 mm layer) and Ma-
cherey&Nagel (RP-18W/UV254, 0.15 mm layer). TLC plates were
colored with vanillin/sulfuric acid (1 g vanillin in 100mL H SO )
Figure 5. Comparison of levels of ROS in U-2 OS cells induced
upon treatment with either tBHP or 4e. I) Control, II) tBHP, and
III) 50μm 4e after 4 h.
2
4
or anisaldehyde (0.50 mL anisaldehyde in 50 mL CH COOH and
3
[34]
1
mL H
2
SO
4
) after heating.
Flash chromatography was per-
formed by using a Teledyne Isco CombiFlash Retrieve chromatog-
raphy separator with commercially available columns (Teledyne
Isco). Column chromatography was performed by using silica gel
60ꢀ (0.04–0.063 mm/230–400 mesh) from Machery&Nagel. Melt-
ing points were determined with a Büchi B-535 or B-545 melting-
Conclusions
The library of naphthoquinones synthesized in this work
showed good antibacterial activities towards Bacillus subti-
lis and Staphylococcus lentus. Naphthoquinones 2c and 2f–
point instrument. NMR spectra were recorded with a Bruker AV-
1
4
00 or Varian V NMR-S 600 spectrometer. H NMR spectra are
referenced to TMS (400 MHz spectra) or solvent signals (all
h exhibited similar MIC values against Staphylococcus au- 600 MHz spectra and all spectra in [D ]DMSO), and 13C NMR
6
reus to the tested reference compounds. Juglonbutin (2o) spectra are referenced to solvent signals. Solvent chemical shifts
showed pronounced activity (2.03 μm) against glycogen syn- used for referencing are: CDCl
3
: δ
H C 6
= 7.26, δ = 77.0 ppm; [D ]-
DMSO: δ = 2.50, δ = 39.5 ppm. EI-MS spectra were recorded
H C
thase kinase 3β, which is a target for the treatment of type 2
diabetes and cancer. Three of our compounds (2f, 2m, and
with a Finnigan MAT-95 spectrometer. The purity of the naphtho-
quinone library (2e–q, 3a, 4a–d) was confirmed by quantitative
microanalyses (C, H, and N); the new compounds were in accord-
ance with the theoretical value to within Ϯ0.4%, that is, a purity
higher than 95%.
2
n) showed very good cytotoxic activity against 12 tumor
cell lines in the lower micromolar range, but grecoketidone
2l), the aglycon of the natural products grecoketides A and
(
B, showed no pronounced cyctotoxic activity, similarly to
1
,5-Diacetoxynaphthalene (1b): A solution of 1,5-dihydroxynaphth-
alene (12.0 g, 74.9 mmol) in acetic acid anhydride (21.0 mL,
23 mmol) and pyridine (134 mL, 1.66 mol) was stirred for 1 h at
5 °C. The reaction mixture was poured onto ice (150 mL) and
structural comparable naphthoquinones, for example, 5-
hydroxylapachol and gonioquinone.[
8,13,14]
Within our li-
2
3
brary of compounds, the cytotoxic activities of the 3-alkyl-
ated naphthoquinones were lower than those of their corre-
sponding 2-O-alkylated analogues (2e in comparison with
ethyl acetate (200 mL) was added. The brown precipitate was fil-
tered and the filtrate was washed with water, saturated CuSO solu-
tion, water, and finally twice with NaCl solution. The organic layer
was dried with Na SO and the solvent was evaporated. Both solids
thoquinones 2f, 2m, and 2n in comparison with the double- were combined and recrystallized from toluene (12.0 g, 66%).
4
2f). The 2-alkoxylated quinones showed the best activities.
The higher cytotoxicities of the 2-monosubstituted naph-
2
4
substituted naphthoquinones 2j, 2q, and 4e is supported by Brown crystals, m.p. 158–159 °C. R
f
= 0.65 (SiO
2
, CHCl
3
/MeOH,
Eur. J. Org. Chem. 2014, 5318–5330
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5323

T. Paululat et al.
FULL PAPER
8
5:15), 0.05 (SiO
2
, CHCl
): δ
J = 7.9, J = 0.5 Hz, 2 H, 2-H, 6-H), 7.50 (t, J = 7.9 Hz, 2 H, 3-
3
); blue stainable with vanillin/H
2
SO
4
. 1H
CDCl
3
): δ
H
= 3.90 (s, 1 H, OCH
3
), 6.08 (s, 1 H, 3-H), 7.24 (dd, J
3
4
3
NMR (400 MHz, CDCl
3
H
= 2.46 (s, 6 H, 2 CO-CH ), 7.28 (dd,
3
= 8.4, J = 1.1 Hz, 1 H, 6-H), 7.56 (dd, J = 8.4, 7.6 Hz, 1 H, 7-
H), 7.64 (dd, J = 7.6, J = 1.1 Hz, 1 H, 8-H), 12.20 (s, 1 H, 5-
OH) ppm. C NMR (100 MHz, CDCl ): δ = 57.1 (q, OCH ),
3 C 3
109.9 (d, C-3), 114.6 (s, C-4a), 120.0 (d, C-8), 125.6 (d, C-6), 131.5
(s, C-8a), 135.9 (d, C-7), 161.5 (s, C-2, C-5), 179.8 (s, C-1), 191.1
3
4
3
3
4
3
4
13
13
H, 7-H), 7.78 (dd, J = 7.9, J = 0.5 Hz, 2 H, 4-H, 8-H) ppm.
NMR (100 MHz, CDCl ): δ = 21.0 (q, 2 CO-CH ), 118.8 (d, C-
, C-6), 119.3 (d, C-4, C-8), 126.0 (d, C-3, C-7), 128.1 (s, C-4a, C-
a), 146.7 (s, C-1, C-5), 169.3 (s, 2 CO-CH ) ppm. C14
C
3
C
3
2
8
3
H
12
O
4
(s, C-4) ppm. C11
64.70, H 3.93.
8 4
H O (204.18): calcd. C 64.71, H 3.95; found C
(244.24): calcd. C 68.85, H 4.95; found C 68.94, H 4.93.
2
,5-Dihydroxynaphthalene-1,4-dione (2-Hydroxyjuglone, 2d): 2-
Hydroxyjuglone (2d) was prepared from 2c (459 mg, 2.20 mmol) by
dissolving 2c in aqueous K CO solution (1 n, 6 mL, 56.0 mmol,
MeOH/H O, 1:1) for 40 min at 80 °C under reflux. After cooling
to room temperature the reaction mixture was diluted with water
45.0 mL) and adjusted to pH 5 with acetic acid. The precipitate
was filtered, washed with water, and recrystallized from 2-propanol
392 mg, 92%). Red crystals, m.p. Ͼ300 °C. R = 0.09 (SiO
CHCl /MeOH, 85:15), 0.34 (SiO , acetone/H O, 1:99); red stain-
able with vanillin/H SO , red stainable with anisaldehyde. H NMR
5-Acetoxy-2-chloronaphthalene-1,4-dione (2a): 5-Acetoxy-2-chloro-
naphthalene-1,4-dione (2a) was prepared from 1,5-diacetoxy-
naphthalene (1b; 3.00 g, 12.3 mmol) by dissolving the latter in
acetic acid (123 mL) at 50 °C and adding a solution of N-chloro-
succinimide (6.58 g, 49.2 mmol) in acetic acid (123 mL) and water
2
3
2
(
(
246 mL) at the same temperature within 1 h. The reaction mixture
was heated at 70–75 °C for 90 min and then poured onto ice
600 mL). The yellow precipitate was filtered and recrystallized
from n-butanol (1.63 g, 53%). Yellow needles, m.p. 140–141 °C. R
0.33 (SiO , CHCl ); blue stainable with vanillin/H SO , yellow
stainable with anisaldehyde. H NMR (400 MHz, CDCl ): δ
(
f
2
,
(
3
2
2
f
1
=
2
3
2
4
2
4
3
4
1
(400 MHz, CDCl ): δH = 6.30 (s, 1 H, 3-H), 7.32 (dd, J = 8.3, J
=
3
3
H
4
3
3
= 0.8 Hz, 1 H, 6-H), 7.58 (dd, J = 8.3, 7.8 Hz, 1 H, 7-H), 7.68
(dd, J = 7.8, J = 0.8 Hz, 1 H, 8-H), 12.32 (s, 1 H, 5-OH) ppm.
C NMR (100 MHz, CDCl ): δ = 110.3 (d, C-3), 114.5 (s, C-4a),
3 C
2
1
7
=
6
1
.44 (s, 3 H, CO-CH
3
), 7.10 (s, 1 H, 3-H), 7.42 (dd, J = 7.9, J =
.3 Hz, 1 H, 6-H), 7.78 (t, J = 7.9 Hz, 1 H, 7-H), 8.13 (dd, J =
3
4
3
3
1
3
4
13
.9, J = 1.3 Hz, 1 H, 8-H) ppm. C NMR (100 MHz, CDCl
21.4 (q, 2 CO-CH ), 123.5 (s, C-4a), 126.5 (d, C-8), 130.8 (d, C-
), 133.3 (s, C-8a), 135.4 (d, C-7), 137.4 (d, C-3), 145.4 (s, C-2),
50.2 (s, C-5), 169.7 (s, CO-CH ), 177.9 (s, C-1), 181.6 (s, C-4) ppm.
3
): δ
C
119.7 (d, C-8), 126.7 (d, C-6), 129.3 (s, C-8a), 135.2 (d, C-7), 156.9
3
(
(
s, C-2), 161.4 (s, C-5), 181.2 (s, C-1), 191.2 (s, C-4) ppm. MS
+
+
EI): m/z (%) = 190 (100) [M] , 162 (26) [M – CO] , 134 (25)
3
+
+
+
8 6 2 10 6 4
[C H O ] , 121 (85). C H O (190.03): calcd. C 63.16, H 3.18;
MS (EI): m/z (%) = 250 (60) [M] , 208 (100) [M – C
2
H
2
O] , 173
+
+
found C 63.35, H 3.18.
(47) [M – C
2 2
H O – Cl] , 145 (10) [M – CO – Cl] , 43 (28).
C
12
H
7
ClO (250.63): calcd. C 57.51, H 2.82; found C 57.62, H 2.76.
4
2
,5-Dihydroxy-3-(ethoxycarbonylmethyl)naphthalene-1,4-dione
(
(
Grecoketidone Ethyl Ester, 2e): A solution of 2-hydroxyjuglone
2d; 1.50 g, 7.89 mmol) in anhydrous DMSO (9.9 mL) under argon
2
-Chloro-5-hydroxynaphthalene-1,4-dione (Chlorojuglone, 2b): 2-
Chloro-5-hydroxynaphthalene-1,4-dione (2b) was synthesized from
was frozen at –78 °C. The frozen surface was covered with LiH
(
(
elimination of H
8
2a (1.00 g, 4.00 mmol) by adding fresh sublimated AlCl
3
(3.00 g,
Cl
2
72.0 mg, 9.06 mmol) and allowed to thaw under inert gas. KI
289 mg, 1.74 mmol) was added to the defrosted solution and after
2
(gas bubbles) ethyl bromoacetate (1.42 g, 943 μL,
.53 mmol) was added. The mixture was stirred at 55 °C for 4 h.
22.5 mmol) portionwise to a stirred solution of 2a in CH
2
(33.0 mL) . The reaction mixture was stirred for 3 h before being
poured onto ice water (150 mL) and stirring for a further 3 h. The
organic layer was extracted with water (3ϫ20.0 mL), dried with
After cooling to room temperature the mixture was poured onto
ice (30 mL). The precipitate was filtered and the filtrate adjusted
Na
obtained after column chromatography (SiO
and recrystallization from 2-propanol (797 mg, 96%). Orange crys-
tals, m.p. 109–110 °C. R = 0.71 (SiO , cyclohexane/ethyl acetate,
0:40), 0.57 (SiO , CHCl ); blue stainable with vanillin/H SO
orange stainable with anisaldehyde. H NMR (400 MHz, CDCl
2
SO
4
, and evaporated under reduced pressure. The product was
2
, 1.5ϫ20 cm, CHCl
3
)
to pH 2 leading to precipitation. Column chromatography (SiO
column 5 ϫ 35 cm, cyclohexane/acetone, 6:4, containing 0.1 %
CH COOH) of the combined precipitates led to two fractions that
were each concentrated in vacuo, diluted in CHCl , and washed
with aqueous NaCl solution and pure water. The organic layers
were dried with Na SO and the solvents evaporated in vacuo.
2
,
f
2
3
6
2
3
2
4
,
1
3
3
):
H
= 7.19 (s, 1 H, 3-H), 7.31 (dd, J = 8.3, J = 1.1 Hz, 1 H, 6-H),
3
4
δ
.64 (dd, 3J = 8.3, 7.5 Hz, 1 H, 7-H), 7.72 (dd, J = 7.5, J =
3
4
2
4
7
1
13
.1 Hz, 1 H, 8-H), 11.78 (s, 1 H, 5-OH) ppm. C NMR (100 MHz,
): δ = 117.0 (s, C-4a), 121.1 (d, C-8), 125.7 (d, C-6), 131.5
s, C-8a), 136.3 (d, C-3), 136.9 (d, C-7), 147.5 (s, C-2), 162.1 (s, C-
2
-(Ethoxycarbonylmethoxy)-5-hydroxynaphthalene-1,4-dione (2f):
Fraction 1 gave 2f, recrystallized from ethanol (562 mg, 26%), m.p.
37–138 °C. R = 0.41 (SiO , cyclohexane/ethyl acetate, 60:40), 0.28
SiO , CHCl ), 0.31 (SiO , CHCl /methanol, 85:15), 0.53 (SiO , cy-
clohexane/acetone, 6:4, with 0.1% CH COOH); orange stainable
with vanillin/H SO , yellow stainable with anisaldehyde. H NMR
600 MHz, CDCl ): δ
J = 7.1 Hz, 2 H, 11-H
CDCl
(
3
C
1
(
f
2
5), 177.7 (s, C-1), 188.3 (s, C-4) ppm. MS (EI): m/z (%) = 208 (100)
2
3
2
3
2
+
+
+
5 3
[M] , 173 (97) [M – Cl] , 145 (35) [M – Cl – CO] . C10H ClO
3
(208.60): calcd. C 57.58, H 2.42; found C 57.73, H 2.37.
1
2
4
3
(
3
H
= 1.30 (t, J = 7.1 Hz, 3 H, 12-H
), 4.71 (s, 2 H, 9-H ), 5.97 (s, 1 H, 3-H),
.24 (dd, J = 8.4, J = 1.1 Hz, 1 H, 6-H), 7.56 (dd, J = 8.4, 7.6 Hz,
3
), 4.28 (q,
5
2
-Hydroxy-2-methoxynaphthalene-1,4-dione
c): To synthesize 2-methoxyjuglone (2c), 2b (1.00 g, 4.80 mmol)
(2-Methoxyjuglone,
3
2
2
3
4
3
7
1
was dissolved in methanol (143 mL) and 2 n NaOH (2.40 mL) was
added dropwise at 40 °C. The reaction mixture was stirred at room
temperature for 2 h. The solvent was evaporated and the solid resi-
3
4
H, 7-H), 7.65 (dd, J = 7.6, J = 1.1 Hz, 1 H, 8-H), 12.06 (s, 1
1
3
H, 5-OH) ppm. C NMR (150 MHz, CDCl
2.2 (t, C-11), 65.6 (t, C-9), 110.8 (d, C-3), 114.2 (s, C-4a), 119.7
d, C-8), 125.3 (d, C-6), 131.1 (s, C-8a), 135.8 (d, C-7), 159.3 (s, C-
), 161.2 (s, C-5), 166.2 (s, C-10), 178.8 (s, C-1), 190.6 (s, C-4) ppm.
3 C
): δ = 14.2 (q, C-12),
6
(
2
due was taken up in CH
The organic layer was dried with Na
rated under vacuum. The crude product was purified by column
chromatography (SiO 5ϫ23 cm, cyclohexane/ethyl acetate,
0:40), leading to 2-methoxyjuglone (2c) as an orange brown solid
0.83 g, 85%), m.p. 164–165 °C. R = 0.41 (SiO , cyclohexane/ethyl
acetate, 60:40), 0.27 (SiO , CHCl ); orange stainable with vanillin/
SO , yellow stainable with anisaldehyde. H NMR (400 MHz, (Grecoketidone Ethyl Ester, 2e): Fraction 2 (2e), was diluted in hot
2
Cl
2
and washed with water (3ϫ50.0 mL).
2
SO and the solvents evapo-
4
+
+
MS (EI): m/z (%) = 276 (53) [M] , 247 (24) [M – C
2
H
5
] , 203 (100)
2
,
+
+
[M – C
3
H
5
O
2
] , 173 (43) [C10
5 4 12 6
H O ] . C14H O (276.24): calcd. C
6
(
6
0.87, H 4.38; found C 60.74, H 4.28.
f
2
2
3
2,5-Dihydroxy-3-(ethoxycarbonylmethyl)naphthalene-1,4-dione
1
H
2
4
5324
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 5318–5330

First Synthesis of Juglonbutin
cyclohexane, filtered, and crystallized from cyclohexane (200 mg,
2,5-Dihydroxy-3-(3-methylbut-2-enyl)naphthalene-1,4-dione
1
9
%). Orange crystals, m.p. 158–160 °C. H NMR (600 MHz,
(Hydroxylapachol, 2g): Fraction 2, recrystallized from cyclohexane
3
[13]
CDCl
3
): δ
H
= 1.28 (t, J = 6.8 Hz, 3 H, 12-H
3
3
), 3.62 (s, 2 H, 9-H
2
), (578 mg, 28 %). Orange needles, m.p. 142–143 °C (ref.
), 7.29 (dd, J = 8.2, J = 1.1 Hz, 145 °C). R = 0.53 (SiO
(SiO , toluene/cyclohexane, 70:30, with 0.1 % CH
stainable with vanillin/H SO , red stainable with anisaldehyde. H
NMR (600 MHz, CDCl , TMS): δ
8.4 (t, C-9), 61.3 (t, C-11), 114.2 (s, C-4a), 116.5 (s, C-3), 119.5 12-H ), 1.78 (s, 3 H, 13-H ), 3.33 (d, J = 7.4 Hz, 2 H, 9-H
144–
3
4
4.19 (q, J = 6.8 Hz, 2 H, 11-H
2
f
2
, cyclohexane/ethyl acetate, 60:40), 0.21
COOH); red
3
3
3
1
H, 6-H), 7.55 (dd, J = 8.2, J = 7.5 Hz, 1 H, 7-H), 7.65 (dd, J
2
3
4
1
=
5
2
7.5, J = 1.1 Hz, 1 H, 8-H), 7.67 (s, 1 H, 2-OH), 12.25 (s, 1 H,
2
4
-OH) ppm. 13C NMR (150 MHz, CDCl
): δ
C
= 14.1 (q, C-12),
4
3
3
H
= 1.70 (d, J = 1.2 Hz, 3 H,
3
3
3
3
2
), 5.19
4
3
4
(d, C-8), 126.5 (d, C-6), 129.4 (s, C-8a), 135.2 (d, C-7), 154.6 (s, C- (tq, J = 7.0, J = 1.2 Hz, 1 H, 10-H), 7.28 (dd, J = 8.4, J =
3
3
5
), 161.4 (s, C-2), 169.7 (s, C-10), 180.4 (s, C-1), 189.8 (s, C-4) ppm.
1.2 Hz, 1 H, 6-H), 7.43 (s, 1 H, 2-OH), 7.53 (dd, J = 8.4, J =
+
+
3
4
MS (EI): m/z (%) = 276 (19) [M] , 246 (26) [M – C
2
H
5
– H] , 231
7.4 Hz, 1 H, 7-H), 7.63 (dd, J = 7.4, J = 1.2 Hz, 1 H, 8-H), 12.50
(s, 1 H, 5-OH) ppm. C NMR (150 MHz, CDCl ): δ = 17.9 (q,
3 C
+
+
+
13
(14) [M – C
3
H
5
O] , 203 (19) [M – C
3
H
5
O
2
] , 174 (38) [C10
H
5
O
3
] .
C
14
H
12
O
6
(276.24): calcd. C 60.87, H 4.38; found C 60.57, H 4.25.
C-13), 22.0 (t, C-9), 25.7 (q, C-12), 114.5 (s, C-4a), 119.2 (d, C-10),
119.3 (d, C-8), 123.1 (s, C-3), 126.2 (d, C-6), 129.4 (s, C-8a), 134.1
R
f
= 0.31 (SiO , CHCl /MeOH, 85:15), 0.30 (SiO , cyclohexane/
2
3
2
acetone, 60:40, with 0.1% CH
SO , orange stainable with anisaldehyde.
-(Ethoxycarbonylmethoxy)-5-hydroxynaphthalene-1,4-dione (2f) as
Single Product: NaOH (2 n, 0.5 mL, 1 mmol) was added dropwise
to a solution of 2-chloro-5-hydroxynaphthalene-1,4-dione (2b;
3
COOH); red stainable with vanillin/ (s, C-11), 134.9 (d, C-7), 153.2 (s, C-2), 161.3 (s, C-5), 180.9 (s, C-
+
H
2
4
1), 190.7 (s, C-4) ppm. MS (EI): m/z (%) = 258 (38) [M] , 243 (100)
+
+
[M – CH
3
] , 215 (9) [M – C
3 7 14 4
H ] . C15H O (258.27): calcd. C
2
69.76, H 5.46; found C 69.61, H 5.23.
2
-(3-Methylbut-2-enyloxy)-5-hydroxynaphthalene-1,4-dione (2h):
2
00 mg, 0.96 mmol) and ethyl glycolate (86.0 μL, 0.90 mmol) in
CH CN. The stirred reaction mixture was heated at 70 °C for 11 h
followed by the addition of NaOH (2 n, 100 μL) and ethyl glycolate
40.0 μL, 0.42 mmol) and further stirring for 13 h at 70 °C. A final
addition of NaOH (2 n, 50 μL) and ethyl glycolate (20.0 μL,
.21 mmol) and stirring for 1 h at 70 °C gave the final reaction
mixture, which was poured onto water. The mixture was extracted
with CHCl . The organic phase was washed with brine and dried
with Na SO . Column chromatography (SiO , column 1ϫ30 cm,
cyclohexane/acetone, 6:4, containing 0.1% CH COOH) led after
crystallization from ethanol to orange needles of 2f (79.5 mg, 30%).
,5-Dihydroxy-3-(3-methylbut-2-enyl)naphthalene-1,4-dione
Hydroxylapachol, 2g): A solution of 2-hydroxyjuglone (2d; 1.50 g,
.89 mmol) in anhydrous DMSO (9.9 mL) under argon was frozen
at –78 °C. The frozen surface was covered with LiH (72.0 mg,
.06 mmol) and was then allowed to thaw under an inert gas. KI
289 mg, 1.74 mmol) was added to the thawed solution and after
the elimination of H (gas bubbles) 1-bromo-3-methyl-2-butene
1.27 g, 933 μL, 8.53 mmol) was added dropwise. The reaction mix-
Fraction 3, recrystallized from methanol (33.1 mg, 2%). Orange
3
crystals, m.p. 139–140 °C, (ref.[ 135–136 °C). H NMR (600 MHz,
8]
1
3
CDCl
3
): δ
H
= 1.76 (s, 3 H, 12-H
3
), 1.81 (s, 3 H, 13-H
3
), 4.60 (d, J
(
3
=
6.8 Hz, 2 H, 9-H
2
), 5.48 (t, J = 6.8 Hz, 1 H, 10-H), 6.08 (s, 1
3
4
3
H, 3-H), 7.26 (dd, J = 8.3, J = 1.1 Hz, 1 H, 6-H), 7.56 (dd, J =
.3, J = 7.6 Hz, 1 H, 7-H), 7.66 (dd, J = 7.6, J = 1.1 Hz, 1 H,
-H), 12.26 (s, 1 H, 5-OH) ppm. C NMR (150 MHz, CDCl
18.4 (q, C-12), 25.8 (q, C-13), 66.7 (t, C-9), 110.0 (d, C-3), 114.2
s, C-4a), 116.9 (d, C-10), 119.5 (d, C-8), 125.0 (d, C-6), 131.1 (s,
C-8a), 135.4 (d, C-7), 140.9 (s, C-11), 160.2 (s, C-2), 161.0 (s, C-5),
0
3
3
4
8
8
1
3
3 C
): δ
3
=
(
2
4
2
3
1
79.6 (s, C-1) 191.0 (s, C-4), 166.2 (s, C-10) ppm. MS (EI): m/z (%)
2
(
7
+
+
+
=
C
R
258 (38) [M] , 243 (74) [M – CH
(258.27): calcd. C 69.76, H 5.46; found C 69.92, H 5.56.
= 0.71 (SiO , cyclohexane/ethyl acetate, 60:40), 0.52 (CHCl ),
, toluene/cyclohexane, 70:30, with 0.1 % CH COOH);
, red stainable with anisal-
3 3 7
] , 215 (13) [M – C H ] .
15
14 4
H O
f
2
3
0
.09 (SiO
2
3
9
(
brown stainable with vanillin/H
dehyde.
2 4
SO
2
(
2
,5-Bis(ethoxycarbonylmethoxy)naphthalene-1,4-dione (2j): A solu-
tion of 2-hydroxyjuglone (2d; 500 mg, 2.63 mmol) in DMF
17.3 mL) was added to a suspension of K CO (727 mg,
.26 mmol) in DMF (10 mL) and the mixture was stirred for
5 min before ethyl bromoacetate (1.77 g, 1.14 mL, 10.5 mmol) was
ture was stirred at 45 °C for 4 h and 15 h at room temperature. The
mixture was poured onto ice and the starting material 2d precipi-
tated and recrystallized from toluene. The filtrate was adjusted to
pH 2 and the new precipitate was filtered. Flash chromatography
(
6
1
2
3
(
40 g SiO
2
column, toluene/cyclohexane, 7:3, with 0.1 %
added dropwise. The reaction mixture was then stirred for 3 d at
room temperature and quenched by pouring onto ice water. The
mixture was extracted with ethyl acetate/pentane (1:1) and the or-
CH COOH) of the precipitate led to three fractions, which were
3
washed with water, dried with Na
evaporated under vacuum.
2 4
SO , filtered, and the solvents
ganic layer was dried with Na
2
SO
column, hexane/ethyl acetate/
, 2:1:1). The major fraction led, after recrystallization from
crystals, m.p. 151–151 °C (ref.[ 123–124 °C). H NMR (600 MHz, ethanol, to 2,5-bis(ethoxycarbonylmethoxy)naphthalene-1,4-dione
4
, evaporated, and purified by
2
,5-Dihydroxy-3-(2-methylbut-3-en-2-yl)naphthalene-1,4-dione (2i): flash chromatography (40 g SiO
2
Fraction 1, recrystallized from n-heptane (167 mg, 8%). Orange CHCl
3
8]
1
3
2
CDCl
3
): δ
H
= 1.57 (s, 6 H, 12-H
3
3
, 13-H
3
), 4.96 (dd, J = 10.6, J
(2j; 89.6 mg, 9 %). Yellow needles, m.p. 118–119 °C. R
(SiO , cyclohexane/acetone, 60:40, with 0.1 % CH COOH). H
NMR (600 MHz, CDCl
f
= 0.39
2
1
=
0.8 Hz, 1 H, 11-H
a
), 5.00 (dd, J = 17.4, J = 0.8 Hz, 1 H, 11-
2
3
3
3
3
3
H
b
), 6.27 (dd, J = 17.4, J = 10.6 Hz, 1 H, 10-H), 7.28 (dd, J =
3
): δ
1.30 (t, J = 7.3 Hz, 3 H, 12-H
H
= 1.28 (t, J = 7.0 Hz, 3 H, 16-H
3
),
),
3
3
3
3
3
7
7
.4, J = 1.1 Hz, 1 H, 6-H), 7.51 (dd, J = 7.5, J = 7.4 Hz, 1 H,
-H), 7.62 (dd, J = 7.5, J = 1.1 Hz, 1 H, 8-H), 7.97 (s, 1 H, 2-
3
), 4.26 (q, J = 7.0 Hz, 2 H, 15-H
4.27 (q, J = 7.3 Hz, 2 H, 11-H ), 4.67 (s, 2 H, 9-H
13-H ), 5.97 (s, 1 H, 3-H), 7.21 (dd, J = 8.4, J = 0.9 Hz, 1 H, 6-
28.4 (q, 2 C, C-12, C-13), 41.0 (s, C-9), 109.8 (s, C-11), 115.2 (s, H), 7.62 (dd, J = 8.3, J = 7.8 Hz, 1 H, 7-H), 7.87 (dd, J = 7.7,
2
3
3
3
2
2
), 4.80 (s, 2 H,
1
3
3
4
OH), 12.55 (s, 1 H, 5-OH) ppm. C NMR (150 MHz, CDCl
=
3
): δ
C
2
3
3
3
4
13
C-4a), 118.9 (d, C-8), 126.7 (d, C-6), 127.5 (d, C-3), 128.5 (s, C-
3 C
J = 1.0 Hz, 1 H, 8-H) ppm. C NMR (150 MHz, CDCl ): δ =
8
1
a), 134.7 (d, C-7), 148.0 (d, C-10), 153.5 (s, C-2), 161.4 (s, C-5),
14.1 (q, C-12, C-16), 61.6 (t, C-11), 62.0 (t, C-15), 65.2 (t, C-9),
66.6 (t, C-13), 113.1 (d, C-3), 120.2 (s, C-4a), 121.0 (d, C-6), 121.1
(d, C-8), 133.4 (s, C-8a), 134.2 (d, C-7), 156.7 (s, C-2), 157.6
(s, C-5), 166.3 (s, C-10), 168.1 (s, C-14), 179.5 (s, C-1), 183.6 (s, C-
81.5 (s, C-1), 191.3 (s, C-4) ppm. MS (EI): m/z (%) = 258 (78)
+
+
+
[
M] , 243 (100) [M – CH
258.27): calcd. C 69.76, H 5.46; found C 69.66, H 5.39. R
SiO , toluene/cyclohexane, 70:30, with 0.1 % CH COOH); red
SO , red stainable with anisaldehyde.
3 3 7 14 4
] , 215 (17) [M – C H ] . C15H O
(
(
f
= 0.37
+
+
2
3
2 6
4) ppm. MS (EI): m/z (%) = 362 (77) [M] , 316 (29) [M – C H O] ,
+
+
stainable with vanillin/H
2
4
289 (59) [M – C
3
H
5
O
2
] , 275 (100) [M – C
4
H
7
O
2
] , 203 (93)
Eur. J. Org. Chem. 2014, 5318–5330
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5325

T. Paululat et al.
FULL PAPER
[
C
10
H
7
O
4
]+, 173 (31) [C10
H
5
O
3
]+. C18
H
18
O
8
(362.33): calcd. C trated in vacuo and dissolved in diethyl ether. Both fractions were
59.67, H 5.01; found C 59.63, H 4.90.
2 4
washed with brine and dried with Na SO .
The aqueous solution was acidified with HCl and extracted with
ethyl acetate. Column chromatography (SiO , hexane/ethyl acetate/
CHCl , 2:1:1) gave grecoketidone ethyl ester (2e), which was filtered
from hot cyclohexane and recrystallized from cyclohexane leading
to pure grecoketidone ethyl ester (2e, 25 mg, 3%, for characteriza-
tion data, see above).
2-[2-(Ethoxycarbonyl)ethoxy]-5-hydroxynaphthalene-1,4-dione (2n):
Fraction 1, recrystallized from cyclohexane (23.4 mg, 80.6 μmol,
2
8%). Yellow needles, m.p. 109–111 °C. R
ane/acetone, 6:4, with 0.1% CH COOH); orange stainable with va-
nillin/H SO , yellow stainable with anisaldehyde. H NMR
(600 MHz, CDCl
f 2
= 0.53 (SiO , cyclohex-
3
3
1
2
4
3
3
): δ
H
= 1.28 (t, J = 7.1 Hz, 3 H, 13-H
3
), 2.92 (t,
), 4.19 (q, J = 7.1 Hz, 2 H, 12-H ), 4.28
2
t, J = 6.3 Hz, 2 H, 9-H ), 6.13 (s, 1 H, 3-H), 7.25 (d, J = 7.3 Hz,
3
3
J = 6.3 Hz, 2 H, 10-H
2
2
2
-(Acetylmethoxy)-5-hydroxynaphthalene-1,4-dione (2l): A solution
of 2-hydroxyjuglone (2d; 20.0 mg, 0.11 mmol) in DMF (2.00 mL)
was added to a suspension of Li CO (8.66 mg, 0.12 mmol) in
3
3
(
1
7
3
3
H, 6-H), 7.55 (dd, J = 7.6, 7.3 Hz, 1 H, 7-H), 7.63 (d, J =
13
2
3
.6 Hz, 1 H, 8-H), 12.18 (s, 1 H, 5-OH) ppm. C NMR (150 MHz,
): δ = 14.2 (q, C-13), 33.5 (t, C-10), 61.3 (t, C-12), 65.1 (t,
C-9), 110.3 (d, C-3), 114.2 (s, C-4a), 119.6 (d, C-8), 125.2 (d, C-6),
31.2 (s, C-4a), 135.6 (d, C-7), 160.2 (s, C-2), 161.2 (s, C-5), 170.1
DMF (4.00 mL) and the mixture was stirred for 15 min before
CDCl
3
C
bromo-2-propanone (27.3 mg, 16.7 μL, synthesized according to
[
35]
the literature ) was added dropwise. The reaction mixture was
stirred for 15 h and quenched by pouring onto ice water. The mix-
ture was extracted with ethyl acetate (2ϫ 15 mL) and the organic
1
(
2
(
s, C-11), 179.1 (s, C-1), 190.9 (s, C-4) ppm. MS (EI): m/z (%) =
+
+
+
90 (100) [M] , 202 (53) [M – C
5
H
10
O
2
] , 190 (25) [C10
H
6
O
4
] , 174
layer was dried with Na
2
SO
4
and removed in vacuo. Recrystalli-
+
25) [C10
H
6
O
3
] . C15
H
14
O
6
(290.27): calcd. C 62.07, H 4.86; found
zation from methanol led to product 2l (7.04 mg, 27%). Brown
C 62.01, H 4.81.
1
needles, m.p. 176–178 °C (decomp.). H NMR (600 MHz, CDCl
3
):
5-[2-(Ethoxycarbonyl)ethoxy]-4-hydroxynaphthalene-1,2-dione (3a):
δ
7
7
H
= 2.34 (s, 3 H, 11-H
.27 (dd, J = 8.3, J = 1.1 Hz, 1 H, 6-H), 7.59 (dd, J = 8.3, J =
3
), 4.66 (s, 2 H, 9-H
2
), 5.93 (s, 1 H, 3-H),
3
4
3
3
Fraction 2, recrystallized from ethanol (12.2 mg, 42.0 μmol, 4%).
Red crystals, m.p. 118–120 °C. R = 0.13 (SiO , cyclohexane/acet-
3
4
.6 Hz, 1 H, 7-H), 7.67 (dd, J = 7.6, J = 1.1 Hz, 1 H, 8-H), 12.09
f
2
s, 1 H, 5-OH) ppm. 1 C NMR (150 MHz, CDCl
3
): δ
C-11), 72.9 (t, C-9), 110.7 (d, C-3), 114.1 (s, C-4a), 119.6 (d, C-8),
25.3 (d, C-6), 131.0 (s, C-8a), 135.7 (d, C-7), 159.0 (s, C-2), 161.1
s, C-5), 178.8 (s, C-1), 190.4 (s, C-4), 201.0 (s, C-10) ppm. MS (EI):
= 26.5 (q,
3 f 2 3
one, 6:4, with 0.1 % CH COOH), R = 0.10 (SiO , CHCl /ethyl
(
3
C
acetate, 6:4); orange stainable with vanillin/H
stainable with anisaldehyde. H NMR (600 MHz, CDCl
2
SO
4
, orange-brown
): δ
2
), 2.95 (t, J = 5.7 Hz, 2 H, 10-H ),
1
3
H
=
1
(
3
3
1
.32 (t, J = 7.2 Hz, 3 H, 13-H
3
3
3
+
+
+
4.26 (q, J = 7.2 Hz, 2 H, 12-H ), 4.49 (t, J = 5.7 Hz, 2 H, 9-H ),
m/z (%) = 246 (30) [M] , 204 (100) [C10
C
R
hexane/acetone, 60:40, with 0.1% CH
with vanillin/H SO
H
7
O
4
] , 173 (21) [C10
(246.22): calcd. C 63.42, H 4.09; found C 63.47, H 4.12.
= 0.24 (SiO , cyclohexane/ethyl acetate, 60:40), 0.45 (SiO , cyclo-
COOH); orange stainable
, yellow stainable with anisaldehyde.
H
5
O
3
] .
2
2
3
3
5
8
.94 (s, 1 H, 3-H), 7.23 (d, J = 8.1 Hz, 1 H, 6-H), 7.45 (dd, J =
.1, 7.5 Hz, 1 H, 7-H), 7.76 (d, J = 7.5 Hz, 1 H, 8-H), 8.82 (s, 1
H, 4-OH) ppm. C NMR (150 MHz, CDCl
3.7 (t, C-10), 62.0 (t, C-12), 65.9 (t, C-9), 103.1 (d, C-3), 114.1 (s,
C-4a), 123.7 (d, C-8), 126.9 (d, C-6), 131.5 (s, C-8a), 133.2 (d, C-
,5-Dihydroxy-3-(carboxymethyl)naphthalene-1,4-dione (Grecoket- 7), 156.4 (s, C-5), 169.9 (s, C-4), 170.5 (s, C-11), 179.2 (s, C-1),
13 10 5
H O
3
f
2
2
1
3
3 C
): δ = 14.3 (q, C-13),
3
3
2
4
2
+
+
idone, 2k): Grecoketidone (2k) was produced after hydrolyzation of
grecoketidone ethyl ester (2e; 61.0 mg, 0.22 mmol) by using a mix-
ture of 2 n NaOH (13.5 mL) and EtOH (6.8 mL) at room tempera-
ture for 40 min. The red reaction mixture was quenched by acidifi-
cation with 2 n HCl. The yellow precipitate was filtered and recrys-
tallized from n-butanol (48.9 mg, 89%). Red-brown crystals, m.p.
179.4 (s, C-2) ppm. MS (ESI ): m/z (%) = 313 (35) [M + Na] , 603
(100) [2M + Na] . C15H14O (290.27): calcd. C 62.07, H 4.86;
+
6
found C 62.52, H 4.85.
2
(
-[3-(Ethoxycarbonyl)propyloxy]-5-hydroxynaphthalene-1,4-dione
2m): Ethyl 4-iodobutanoate (416 mg, 1.72 mmol) was added drop-
wise to a suspension of 2-hydroxyjuglone (2d; 200 mg, 1.06 mmol)
and Li CO (74.0 mg, 1.06 mmol) in DMF (40 mL). The reaction
1
80 °C (decomp.). R
f
= 0.23 (RP, 2-propanol/water, 80:20); red
SO , orange stainable with anisaldehyde.
H 2
]DMSO): δ = 3.41 (s, 2 H, 9-H ), 7.34
2
3
stainable with vanillin/H
2
4
1
mixture was stirred for 17 d, and during days 10 and 13 further
ethyl 4-iodobutanoate (138 mg, 0.57 mmol, synthesized according
to the literature[ ) was added. The reaction mixture was finally
poured onto water and acidified with HCl (pH 5) and extracted
H NMR (400 MHz, [D
6
dd, 3J = 8.3, 4J = 1.0 Hz, 1 H, 6-H), 7.57 (dd, J = 7.4, J =
3
4
(
35]
3
1
1
.0 Hz, 1 H, 8-H), 7.68 (dd, J = 8.3, 7.4 Hz, 1 H, 7-H), 12.38 (s,
H, 5-OH) ppm. 1 C NMR (100 MHz, [D
3
]DMSO): δ = 28.3 (t,
6 C
with CHCl
Na SO , concentrated in vacuo, and subjected to column
chromatography (SiO , cyclohexane/ethyl acetate, 8:2). The product
was recrystallized from cyclohexane (142 mg, 0.47 mmol, 44%).
Yellow needles, m.p. 93–94 °C. R = 0.55 (SiO , cyclohexane/acet-
one, 6:4, with 0.1% CH COOH); orange stainable with vanillin/
3
. The organic layer was washed with brine, dried with
C-9), 114.0 (s, C-4a), 117.5 (s, C-3), 119.0 (d, C-8), 125.2 (d, C-6),
30.5 (s, C-8a), 135.6 (d, C-7), 157.6 (s, C-2), 160.2 (s, C-5), 171.4
s, C-10), 180.5 (s, C-1), 190.9 (s, C-4) ppm. MS (EI): m/z (%) =
2
4
1
2
(
2
+
+
48 (12) [M] , 204 (100) [M – CO
2 8 6
] . C12H O (248.19): calcd. C
f
2
58.07, H 3.25; found C 58.56, H 3.28.
3
1
2
Ag
-[2-(Ethoxycarbonyl)ethoxy]-5-hydroxynaphthalene-1,4-dione (2n):
O (0.92 g, 3.97 mmol) was added in portions and under vigor-
ous stirring to a solution of 2-hydroxyjuglone (2d; 200 mg,
.06 mmol) in CHCl (120 mL), followed by ethyl 3-iodopropano-
ate (280 μL, 479 mg, 2.10 mmol, synthesized according to the lit-
H
CDCl
2
SO
4
, yellow stainable with anisaldehyde. H NMR (600 MHz,
3
3
2
3
): δ
H
= 1.26 (t, J = 7.1 Hz, 3 H, 14-H
3
), 2.21 (tt, J = 6.8,
3
3
6.4 Hz, 2 H, 10-H
= 6.4 Hz, 4 H, 2 H, 9-H
2
), 2.54 (t, J = 6.8 Hz, 2 H, 11-H
2
), 4.08 (t, J
3
1
3
2
), 4.15 (q, J = 7.1 Hz, 2 H, 13-H
2
), 6.09
3
3
(s, 1 H, 3-H), 7.25 (d, J = 8.1 Hz, 1 H, 6-H), 7.56 (dd, J = 8.1,
7.6 Hz, 1 H, 7-H), 7.64 (d, J = 7.6 Hz, 1 H, 8-H), 12.2 (s, 1 H, 5-
O (243 mg, 1.05 mmol) added and the mixture was OH) ppm. C NMR (150 MHz, CDCl
(t, C-10), 30.4 (t, C-11), 60.8 (t, C-13), 68.7 (t, C-9), 110.1 (d, C-
3), 114.4 (s, C-4a), 119.6 (d, C-8), 125.2 (d, C-6), 131.2 (s , C-8a),
[
35]
3
erature ). The mixture was heated under reflux for 2 d, and then
further Ag
heated at reflux for 3 h. The reaction was monitored by TLC (SiO
cyclohexane/acetone, 6:4, with 0.1 % CH COOH). The reaction
mixture was filtered through Celite (ca. 3 g) and concentrated in
vacuo. Column chromatography (SiO , cyclohexane/acetone, 6:4,
with 0.1% CH COOH) gave two fractions, which were concen-
1
3
2
3 C
): δ = 14.3 (q, C-14), 23.7
2
,
3
q
135.6 (d, C-7), 160.4 (s, C-2), 161.2 (s, C-5), 172.7 (s, C-12), 179.3
+
2
(s, C-1), 191.0 (s, C-4) ppm. MS (EI): m/z (%) = 304 (4) [M] , 259
+
+
+
3
(11) [M – C
2
H
5
O] , 231 (10) [C10
H
7
O
4
] , 190 (14) [C10
6 4
H O ] . MS
5326
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 5318–5330

First Synthesis of Juglonbutin
+
+
–
–
(
3
5
ESI ): m/z (%): 327 (100) [M + Na] ; (ESI ): 303 (30) [M – H] , 38%), m.p. 134–135 °C. R
f
= 0.37 (SiO
2
, cyclohexane/acetone, 6:4,
SO , yel-
low stainable with anisaldehyde. H NMR (600 MHz, CDCl ): δ
), 4.27 (q,
), 6.10 (s, 1 H, 3-H),
–
63 (10) [M + CH
.30; found C 63.13, H 5.26.
3
COO] . C16
H
16
O
6
(304.30): calcd. C 63.15, H
with 0.1% CH
3
COOH); orange stainable with vanillin/H
2
4
1
3
H
3
=
1.29 (t, J = 7.2 Hz, 3 H, 13-H
3
), 3.86 (s, 3 H, 9-OCH
), 4.80 (s, 2 H, 10-H
.21 (dd, J = 8.5, J = 0.8 Hz, 1 H, 6-H), 7.61 (dd, J = 8.5, 7.9 Hz,
3
2
-(3-Carboxypropylamino)-5-hydroxynaphthalene-1,4-dione (Juglon-
butin, 2o): A solution of 4-aminobutyric acid (198 mg, 1.92 mmol)
in water (1.92 mL) was added dropwise whilst stirring to a solution
3
of 2-chlorojuglone (2b; 200 mg, 0.96 mmol) in CH CN (20 mL).
The reaction mixture was heated at reflux for 8 h. After cooling,
the solvents were removed under vacuum and the residue purified
by column chromatography (SiO
.1% CH COOH). The two collected fractions were concentrated
in vacuo, diluted with ethyl acetate, and washed with brine. The
3
J = 7.2 Hz, 2 H, 12-H
2
2
3
4
3
7
1
3
4
13
H, 7-H), 7.86 (dd, J = 7.9, J = 0.8 Hz, 1 H, 8-H) ppm.
): δ = 14.4 (q, C-13), 56.5 (q, C-9), 61.8
t, C-12), 67.0 (t, C-10), 112.2 (s, C-3), 120.6 (d, C-6), 121.2 (d,
C-8), 121.4 (s, C-4a), 133.6 (s, C-8a), 134.2 (d, C-7), 157.7 (s, C-5),
58.7 (s, C-2), 168.4 (s, C-11), 180.3 (s, C-1), 184.2 (s, C-4) ppm.
C
NMR (150 MHz, CDCl
3
C
(
2 3
, ethyl acetate/CHCl , 1:1, with
1
0
3
+
+
MS (EI): m/z (%) = 290 (22) [M] , 261 (27) [M – C
2
H
5
] , 217 (100)
+
[M – C
H O ] . C15H O (290.27): calcd. C 62.07, H 4.86; found
3 5 2 14 6
organic layers were dried with anhydrous Na
removed under vacuo.
2 4
SO and the solvent
C 61.79, H 4.65.
5
2
,8-Dimethoxynaphthalene-1,4-dione
.20 mmol) was added portionwise to a solution of 5,8-dihydroxy-
(40 mL),
(4b):
Ag
2
O
(500 mg,
4
-[(2-Chloro-5-hydroxy-1,4-dioxo-3-naphthylamino]butanoic Acid
(
2p): Fraction 1, crystallized from 2-propanol (81.2 mg,
1
1,4-naphthoquinone (4a; 546 mg, 2.90 mmol) in CHCl
followed by the dropwise addition of methyl iodide (2.0 mL,
32.1 mmol). The reaction mixture was heated to 60–65 °C for 4 d,
3
0.26 mmol).Redneedles,m.p.173–178 °C. HNMR(600 MHz,[D
6
]-
), 2.29 (t, J =
), 7.22 (d,
3
3
DMSO): δ
7
H
= 1.85 (tt, J = 7.3, 7.0 Hz, 2 H, 10-H
2
3
.3 Hz, 2 H, 11-H
2
), 3.77 (dt, J = 7.0, 6.6 Hz, 2 H, 9-H
2
3
3
during which time further Ag
methyl iodide (3ϫ1.5 mL, 72.5 mmol) were added. TLC-analysis
(SiO , cyclohexane/CHCl /methanol, 7:2:1). After completion of
the reaction, the mixture was filtered through Celite (4 g) and
washed with CHCl The filtrate was concentrated in vacuo
474 mbar, 40 °C) and the orange residue was crystallized from
nBuOH as orange needles of 4b (430 mg, 1.97 mmol, 69%), m.p.
2
O (4ϫ500 mg, 8.80 mmol) and
J = 8.0 Hz, 1 H, 7-H), 7.49 (br. s, 1 H, 3-NH), 7.51 (d, J = 7.8 Hz,
3
1
H, 8-H), 7.70 (dd, J = 8.0, 7.8 Hz, 1 H, 7-H), 11.3 (br. s, 1 H,
13
2
3
1
2-COOH), 12.0 (br. s, 1 H, 5-OH) ppm. C NMR (150 MHz, [D
DMSO): δ = 26.0 (t, C-10), 30.7 (t, C-11), 43.2 (t, C-9), 113.6 (s,
C-4a), 118.3 (d, C-8), 122.3 (d, C-6), 132.4 (s, C-8a), 137.1 (d, C-
6
]-
C
3
(
7
1
), 144.9 (s, C-3), 145.0 (s, C-2), 160.6 (s, C-5), 174.0 (s, C-12),
–
75.0 (s, C-1), 183.5 (s, C-4) ppm. MS (ESI ): m/z (%) = 308 (90)
157–159 °C (ref.[ 155 °C). R
36]
= 0.16 (SiO
, cyclohexane/CHCl
SO , brown stain-
able with anisaldehyde. H NMR (400 MHz, CDCl ): δ = 3.96 (s,
), 6.78 (s, 2 H, 2-H, 3-H), 7.32 (s, 2 H,
-H, 7-H) ppm. C NMR (100 MHz, CDCl ): δ = 57.0 (q, 5-
, 8-OCH ), 120.5 (s, C-4a, C-8a), 138.5 (d, C-2, C-3, C-6, C-
), 153.8 (s, C-5, C-8), 185.0 (s, C-1, C-4) ppm. MS (EI): m/z (%)
–
+
+
+
f
2
3
/
[M – H] ; (ESI ): 310 (36) [M + H] , 332 (56) [M + Na] , 366 (12)
+
methanol, 7:2:1); violet stainable with vanillin/H
2
4
[
M + K + H
2
O] . C14
H
12ClNO
5
(309.70): calcd. C 54.29, H 3.91,
= 0.22 (SiO , ethyl
COOH); red stainable with va-
4
, brown stainable with anisaldehyde.
1
3
H
N 4.52; found C 54.66, H 3.57, N 4.57. R
acetate/CHCl , 1:1, with 0.1% CH
nillin/H SO
f
2
6
6
3 3 3
H, CH , 5-OCH , 8-OCH
3
3
1
3
3
C
2
OCH
7
=
C
3
3
4
-(5-Hydroxy-1,4-dioxo-2-naphthylamino)butanoic Acid (Juglon-
butin, 2o): Fraction 2 crystallized from n-butanol (50.8 mg,
1
2
CH
anisaldehyde. H NMR (600 MHz, [D
+
+
+
218 (100) [M] , 189 (37) [C10
5 4 4 3
H O ] , 172 (41) [C10H O ] .
85 mmol). Red crystal squares, m.p. 228–230 °C (ref.[ 228–
30 °C). R = 0.15 (SiO , ethyl acetate/CHCl , 1:1, with 0.1 %
COOH); pink stainable with vanillin/H SO , red stainable with
]DMSO): δ
), 2.31 (t, J = 7.2 Hz, 2 H, 11-H
16]
12
10 4
H O
(218.21): calcd. C 66.05, H 4.62; found C 66.70, H 4.68.
f
2
3
2
,5,8-Trimethoxynaphthalene-1,4-dione (4c):
A
suspension of
3
2
4
1
3
H SO (conc., 3.0 mL) and Fe (SO ) (800 mg, 0.2 mmol) was
= 1.80 (tt, J =
), 3.24 (dt,
2
4
2
4 3
6
H
3
added to a solution of 5,8-dimethoxy-1,4-naphthoquinone (4b;
218 mg, 100 μmol) in dioxane (30 mL) and methanol (15 mL), and
the reaction mixture was heated at reflux for 2 h. The reaction was
monitored by TLC (SiO , CHCl /ethyl acetate/methanol, 6:3:1).
7
.2, 6.9 Hz, 2 H, 10-H
2
2
3
3
2
J = 6.9, 6.4 Hz, 2 H, 9-H ), 5.68 (s, 1 H, 3-H), 7.25 (d, J = 8.0 Hz,
H, 6-H), 7.49 (d, 3J = 7.5 Hz, 1 H, 8-H), 7.56 (dd, J = 8.0,
3
1
7
3
.5 Hz, 1 H, 7-H), 8.00 (t, J = 6.4 Hz, 1 H, 2-NH), 12.08 (br. s, 1
2
3
13
The reaction mixture was poured onto water (100 mL), extracted
with CHCl , and dried with Na SO . The solvent was evaporated
474 mbar, 40 °C) and the residue purified by column chromatog-
raphy (SiO , 3.0ϫ30 cm, CHCl /ethyl acetate/methanol, 6:3:1).
H, 12-OH), 13.41 (s, 1 H, 5-OH) ppm. C NMR (150 MHz, [D
DMSO): δ = 22.6 (t, C-10), 30.8 (t, C-11), 41.4 (t, C-9), 98.0 (d,
C-3), 114.4 (s, C-4a), 118 (d, C-8), 125.0 (d, C-6), 130 (s, C-8a),
34.0 (d, C-7), 149.7 (s, C-2), 160.1 (s, C-6), 174.0 (s, C-12), 180.6
6
]-
3
2
4
C
(
1
2
3
–
Two fractions were obtained, the second one containing 2,5,8-tri-
methoxy-1,4-naphthoquinone (4c), which gave orange-colored
crystals following crystallization from toluene (248 mg, 1.06 mmol,
(s, C-1), 187.8 (s, C-4) ppm. MS (ESI ): m/z (%) = 274 (100) [M –
–
+
+
+
H] ; (ESI ): m/z (%) = 276 (60) [M + H] , 298 (44) [M + Na] , 314
+
+
(12) [M + K] , 332 (22) [M + K·H
2 5
O] . C14H13NO (275.26): calcd.
6%), m.p. 173–174 °C (ref.[
CDCl ): δ = 3.82 (s, 3 H, 2-OCH
OCH ), 6.00 (s, 1 H, 3-H), 7.25 (d, J = 9.4 Hz, 1 H, 7-H), 7.32 (d,
37]
165 °C). H NMR (600 MHz,
), 3.94 (s, 6 H, 5-OCH , 8-
1
4
C 61.09, H 4.76, N 5.09; found C 61.53, H 4.77, N 5.06.
3
H
3
3
2
-Methoxy-5-(ethoxycarbonylmethoxy)naphthalene-1,4-dione (2q):
3
3
2-Methoxyjuglone (2c; 300 mg, 1.47 mmol) was dissolved in a sus-
3
13
3 C
J = 9.4 Hz, 1 H, 6-H) ppm. C NMR (150 MHz, CDCl ): δ =
pension of DMF (17.3 mL) and K
2
CO
3
(483 mg, 1.47 mmol).
[a]
[a]
5
(
6.4 (q, 2-OCH
s, C-3), 119.8 (s, C-7), 120.5 (d, C-8a), 121.0 (s, C-4a), 121.4 (d,
C-6), 153.5 (s, C-5), 154.5 (s, C-8), 159.4 (s, C-2), 179.5 (s, C-1),
3 3 3
), 57.0 (q, 8-OCH ), 57.1 (q, 5-OCH ), 110.2
Whilst stirring, ethyl bromoacetate (315 μL, 2.94 mmol) was added
and the reaction mixture was stirred for a further 20 h, poured onto
ice (50 mL) and the pH value was adjusted from pH 11 to pH 6
with HCl (2 n, 1 mL). The yellow precipitate was dissolved by ad-
dition of ethyl acetate. The organic layer was separated, dried with
+
1
84.9 (s, C-4) ppm. MS (EI): m/z (%) = 248 (100) [M] , 233 (60)
+
[M – CH
3
] . C13
3.11, H 4.89. R
:3:1); violet stainable with vanillin/H
H
12
O
5
(248.23): calcd. C 62.90, H 4.87; found C
= 0.59 (SiO , CHCl /ethyl acetate/methanol,
SO , brown stainable with
6
6
f
2
3
Na
2
4
SO , and concentrated in vacuo. Column chromatography
2
4
(SiO
2
, 3ϫ 25 cm, pentane/ethyl acetate, 1:1) led to two fractions.
[a]
anisaldehyde. Signals are changeable.
The first fraction contained 2-methoxyjuglone (2c; 12.5 mg) and
the second fraction was that of the product 2q, which was obtained,
after crystallization from methanol, as yellow crystals (161 mg,
5,8-Dihydroxy-2-methoxynaphthalene-1,4-dione (4d): AgO (1.20 g,
9.69 mmol) was added portionwise whilst stirring to a solution of
Eur. J. Org. Chem. 2014, 5318–5330
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5327

T. Paululat et al.
FULL PAPER
2,5,8-trimethoxy-1,4-naphthoquinone (4c; 217 mg, 0.87 mmol) in
brief, human tumor cell lines were grown at 37 °C in a humidified
acetone (34 mL). After cooling to 0 °C, HNO
3
2
(11.3 mL, 40%) was atmosphere (95% air, 5% CO ) in monolayer cultures in RPMI
added dropwise. The reaction mixture was stirred for 15 min at
room temp., diluted with water (1:2), and finally extracted with
1640 medium supplemented with 10% FCS and Phenol Red (PAA,
Cölbe, Gemany). Cells were trypsinized and maintained weekly.
They were harvested from exponentially growing cultures by tryp-
sination, counted, and plated in 96-well flat-bottomed microplates
(140 μL cell suspension, 5ϫ10 to 10ϫ10 cells/well). After a 24 h
recovery to allow cells to resume exponential growth, 10 μL of cul-
ture medium (6 control wells per plate) or medium containing the
test drug were added to the wells. Each drug concentration was
plated in triplicate. After 4 d of incubation the culture medium was
replaced by a fresh medium containing 6 μg/mL of propidium iod-
CHCl
2ϫ), and dried with Na
residue purified by column chromatography (SiO
CHCl /ethyl acetate, 6:4). The product was obtained after
recrystallization from 2-propanol. Black needles (52.0 mg,
36 μmol, 27%), m.p. 192–193 °C (ref.[37] 190–195 °C). R
= 0.60
COOH), 0.75
/ethyl acetate, 6:4); pink stainable with vanillin/
3
(3ϫ). The organic layer was washed with brine and water
SO . The solvent was evaporated and the
, 1ϫ20 cm,
(
2
4
3
3
2
3
2
f
(
(
SiO
SiO
2
, cyclohexane/acetone, 6:4, with 0.1% CH
, CHCl
3
2
3
1
H
CDCl
=
8
2
SO
4
, red stainable with anisaldehyde. H NMR (600 MHz, ide. Microplates were then kept at –18 °C for 24 h to determine
3
3
): δ
H
= 3.92 (s, 3 H, 2-OCH
3
), 6.16 (s, 1 H, 3-H), 7.19 (d, J
the total cell kill. After thawing of the plates, the fluorescence was
measured by using the Cytofluor 4000 microplate reader (Persep-
tive Biosystems; excitation 530 nm, emission 620 nm). The number
of viable cells was proportional to the fluorescence intensity.
3
9.4 Hz, 1 H, 7-H), 7.26 (d, J = 9.4 Hz, 1 H, 6-H), 12.2 (s, 1 H,
-OH), 12.6 (s, 1 H, 5-OH) ppm. 13C NMR (150 MHz, CDCl
): δ
), 110.4 (s, C-3), 110.6 (s, C-4a), 111.5 (s, C-8a),
3
C
=
56.9 (q, 2-OCH
3
1
28.5 (d, C-7), 131.0 (d, C-6), 157.1 (d, C-5), 158.6 (s, C-8), 161.1
Mechanisms of Action
(
(
s, C-2), 182.6 (s, C-4), 188.4 (s, C-1) ppm. MS (EI): m/z (%) = 220
100) [M] , 202 (29) [M – H O] . C11H O (220.18): calcd. C 60.00,
2 8 5
+
+
Cell Cultures: The cell lines HCT-116 (human colon carcinoma)
and U-2 OS (human osteosarcoma) were obtained from the Ger-
man Collection of Microorganisms and Cell Cultures (DSMZ) and
were cultured under the conditions recommended by the depositor.
Reagents were purchased from Sigma–Aldrich and plastic ware was
obtained from Nunc, BD Falcon, and Sarstedt AG & Co.
H 3.66; found C 59.39, H 3.63.
,8-Bis(ethoxycarbonylmethoxy)-2-methoxynaphthalene-1,4-dione
4e): Ethyl iodoacetate (85.0 μL, 155 mg, 0.72 mmol) was added to
a suspension of 5,8-dihydroxy-2-methoxy-1,4-naphthoquinone (4d;
8.2 mg, 0.13 mmol) and K CO (45.3 mg, 0.33 mmol) in DMF
3.0 mL). The reaction mixture was stirred at room temp. for 3 d
before further K CO (11.3 mg, 0.82 mmol) was added and stirring
5
(
2
(
2
3
3
MTT Assay: Cells were seeded at 6ϫ10 cells per well in 96-well
plates in 180 μL complete medium and directly treated with varying
concentrations of 2f or 2j diluted in MeOH. The compounds were
tested in duplicate as well as the internal vesicle control. After treat-
ment for 5 d, 20 μL of 5 mg/mL MTT (Thiazolyl Blue tetrazolium
bromide) in PBS was added per well and further incubated for 2 h
at 37 °C. The medium was then discarded and cells were washed
with 100 μL PBS (phosphate-buffered saline, pH 7.4) before adding
2
3
was continued for an additional 2 d at the same temperature. The
reaction mixture was then diluted with water (30 mL) and extracted
with CHCl
with Na SO
purified by column chromatography (Combi Flash retrieve, 40 g
SiO column, CHCl /ethyl acetate, 6:4) to give, after crystallization,
orange crystals (17.5 mg, 44.6 μmol, 25%), m.p. 74–76 °C. R
.53 (SiO , cyclohexane/acetone, 6:4, with 0.1% CH COOH), 0.43
SiO , CHCl /ethyl acetate, 6:4); grey-brown stainable with vanillin/
SO
3
. The organic layer was washed with brine and dried
2
4
. After evaporation of the solvent the product was
2
3
100 μL 2-propanol/10 n HCl (250:1) in order to dissolve formazan
f
=
granules. The absorbance at 570 nm was measured by using a
microplate reader (EL808, BioTek Instruments Inc.) and cell viabil-
ity was expressed as a percentage relative to the respective methanol
control.
0
(
H
2
3
2
3
1
2
4
, orange-brown stainable with anisaldehyde. H NMR
3
(
600 MHz, CDCl
.83 (s, 3 H, 9-H
s, 4 H, 10-H , 14-H
H, 6-H), 7.29 (d, J = 9.3 Hz, 1 H, 7-H) ppm. C NMR (150 MHz,
CDCl ): δ = 14.3 (q, C-13, C-17), 56.4 (q, 2-OCH ), 61.6 (t, C-
2, C-16), 68.0 (t, C-10), 68.3 (t, C-14), 110.2 (d, C-3), 122.1 (s, C-
3
): δ
H
= 1.28 (t, J = 7.0 Hz, 6 H, 13-H
3
, 17-H
3
),
3
5
3
(
3
), 4.25 (q, J = 7.0 Hz, 4 H, 12-H
2
3
, 16-H
2
), 4.71
Cell Cycle Analysis: Cells were seeded at 10 cells per well in 6-well
plates in 5 mL complete medium. After a 12 h recovery period, cells
were treated for 3 d with varying concentrations of 2f or 2j diluted
in MeOH. Approximately 10 cells were harvested by centrifuga-
tion, washed with ice-cooled PBS, and fixed overnight at –20 °C
with 80% MeOH. After fixation, MeOH was removed completely
and the cell pellet was washed first with PBS and then with 0.1%
saponine in PBS. For DNA staining, cells were incubated for
2
2
), 6.02 (s, 1 H, 3-H), 7.21 (d, J = 9.3 Hz, 1
3
13
6
3
C
3
1
8
1
a), 122.8 (s, C-4a), 123.4 (s, C-7), 125.5 (s, C-6), 152.9 (s, C-8),
53.9 (s, C-5), 159.5 (s, C-2), 168.6 (s, C-11), 168.8 (s, C-15), 179.0
+
(s, C-1), 184.1 (s, C-4) ppm. MS (EI): m/z (%) = 392 (68) [M] , 319
+
+
+
(85) [M – C
3
H
5
O
2
] , 305 (100) [M – C
4
H
7
O
2
] , 245 (78) [C12
H
5
O
6
] , 30 min at 37 °C in a PBS solution containing 20 μg/mL propidium
+
1
89 (17) [C10
H
5
O
4
] . C19
H
20
O
9
(392.36): calcd. C 58.16, H 5.14;
iodide and 100 μg/mL RNase A. The samples were analyzed by a
flowcytometric system (EasyCyte Plus, Guava Technologies). In
total, 5000 viable cells were acquired per sample and cell cycle his-
tograms were generated after exclusion of cell debris using a Wat-
son algorithm of FlowJo 7.2.6 software (Tree Star Inc.).
found C 58.12, H 5.07.
Biological Assays
Primary Activity Profiling: The results and experimental procedures
for the antibacterial agar diffusion assays, enzyme assays and three
cell line assays are given in the Supporting Information.
3
Immunofluorescence: U-2 OS cells were seeded at 5ϫ10 cells per
well in 96-well imaging plates (BD Falcon). At approximately 70%
confluency the cells were treated with 2f or 2j at the given concen-
trations and treatment periods indicated for each experiment. Cells
were fixed with cold (–20 °C) acetone/MeOH (1:1) for 10 min. Af-
ter washing with PBS, cells were permeabilized by using 0.01%
Triton-X 100 in PBS. The following combinations of primary and
secondary antibodies were used: α-tubulin mAb (1:2000, Sigma)/
goat-anti-mouse-Alexa488 (1:1000, Molecular Probes); β-actin
mAb (1:2000, Cell Signaling)/goat-anti-rabbit-Alexa488 (1:1000,
Molecular Probes); phospho-Histone γH2A·X (Ser139) mAb
In Vitro Antitumor Activity Assays: A modified propidium iodide
assay was used to determine the cytotoxic activity of the com-
pounds against human tumor cell lines. The test procedure has
been described elsewhere.[ The cell lines tested were derived from
patient tumors engrafted as a subcutaneously growing tumor in
NMR I nu/nu mice or obtained from the American Type Culture
Collection, Rockville, MD, USA, the National Cancer Institute,
Bethesda, MD, USA, or the Deutsche Sammlung von Mikroor-
ganismen und Zellkulturen (DSMZ), Braunschweig, Germany. In
29]
5328
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 5318–5330

First Synthesis of Juglonbutin
1:400, Millipore)/goat-anti-mouse-Alexa488 (1:1000, Molecular
Probes). For labelling, cells were incubated with a primary anti-
body for 45 min at 37 °C, followed by a secondary antibody under
the same incubation conditions. The nuclei were stained with
(
Acknowledgments
The authors thank Dr. Erwin Röcker, University of Giessen, for
recording EI mass spectra, Heinrich Bodenstedt, University of
Siegen, for elemental analysis, Arlette Erhard, IFM-GEOMAR,
Kiel, Germany, for her very helpful assistance in conducting the
bioassays. We also thank Prof. Dr. Heiko Ihmels for his continous
support and fruitfull discussions, and the Siegener Studienstiftung
for financial support.
Hoechst33342 (5 μg/mL; Molecular Probes) and a whole cell stain
TM
(1 μg/mL HCS CellMask
Red stain, Invitrogen) was used to fa-
cilitate later cell segmentation. The samples were imaged on an
automated microscope (BD Pathway855) suitable for high-content
screening with appropriate filter sets for Alexa488, Hoechst, and
rhodamine.
Level of Reactive Oxygen Species (ROS): The antioxidative poten-
tial of naphthoquinones was evaluated by a counter-screening ap-
proach by using a fluorescence-based assay. Reduced, acetylated
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hydrofluorescein (DCF), which can be oxidized by reactive oxygen
species (ROS) to the fluorescent 2Ј,7Ј-dichlorofluorescein (DCF).
The concentration of DCF generated is directly proportional to the
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10% FBS) and incubated for 2 d at 37 °C and 5% CO
were washed with KRH buffer (119 mm NaCl, 4.7 mm KCl, 2.5 mm
CaCl , 1.2 mm MgCl , 1.2 mm KH PO , 10mm HEPES, 2 mm d-
glucose; pH 7.4) and incubated with 10 μm DCFDA in KRH for
.5 h at 37 °C. Next, the cells were washed twice with KRH buffer
2
. The cells
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Topoisomerase II Assay: The assay for the determination of the in-
hibitory activity of 2c, 2f, 2g, and 2j was performed according to
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conversion of supercoiled DNA to relaxed DNA by the topoiso-
merase II activity. Purified Human DNA Topoisomerase IIa (p170
form) was obtained from TopoGEN and supercoiled pBR322 plas-
mid DNA was purchased from Fermentas GmbH. The master-mix
contained 50 mm Tris HCl (pH 8), 120 mm KCl, 10 mm
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[
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Received: March 17, 2014
Published Online: July 15, 2014
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