Anti-tumoral activities of dioncoquinones B and C and related naphthoquinones gained from total synthesis or isolation from plants

Article abstract of DOI:10.1016/j.ejmech.2011.09.012

Dioncoquinones belong to a family of natural naphthoquinone products of interest due to their promising anti-tumoral and anti-infective activities. In particular, dioncoquinones A (5) and B (6) have been shown to be highly active against Leishmania major and multiple myeloma cells without any significant toxicity toward normal blood cells. Their effective concentrations against multiple myeloma cell lines were similar to those of melphalan, a well known DNA-alkylating agent used in a standard therapy against B cell lymphoma and multiple myeloma. We report on the first total synthesis of the highly oxygenated anti-tumoral agent dioncoquinone B (6) and the isolation of its new, even higher-oxygenated analogs, dioncoquinones C (7), D (8), and E (9), from cell cultures of Triphyophyllum peltatum. In addition, several derivatives of these compounds were synthesized, including dioncoquinone C (7), and a small library of naphthoquinones was created. Furthermore, the first structure-activity relationship (SAR) study on this class of compounds was conducted, showing that each of the three hydroxy groups, at C-3, C-5, and C-6, is required for improved anti-tumoral activities and decreased cytotoxicities.

Full text of DOI:10.1016/j.ejmech.2011.09.012

European Journal of Medicinal Chemistry 46 (2011) 5778e5789
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anti-tumoral activities of dioncoquinones B and C and related naphthoquinones
gained from total synthesis or isolation from plants
,
a
a
,
1
a
a
Gerhard Bringmanna , Guoliang Zhang , Anastasia Hager , Michael Moos , Andreas Irmer ,
*
Ralf Bargoub, Manik Chatterjeeb
a Institute of Organic Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany
b Department of Internal Medicine II, Comprehensive Cancer Center Mainfranken, Hospital of Würzburg, Versbacher Straße 5, D-97078 Würzburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Dioncoquinones belong to a family of natural naphthoquinone products of interest due to their prom-
ising anti-tumoral and anti-infective activities. In particular, dioncoquinones A (5) and B (6) have been
shown to be highly active against Leishmania major and multiple myeloma cells without any significant
toxicity toward normal blood cells. Their effective concentrations against multiple myeloma cell lines
were similar to those of melphalan, a well known DNA-alkylating agent used in a standard therapy
against B cell lymphoma and multiple myeloma. We report on the first total synthesis of the highly
oxygenated anti-tumoral agent dioncoquinone B (6) and the isolation of its new, even higher-oxygenated
analogs, dioncoquinones C (7), D (8), and E (9), from cell cultures of Triphyophyllum peltatum. In addition,
several derivatives of these compounds were synthesized, including dioncoquinone C (7), and a small
library of naphthoquinones was created. Furthermore, the first structure-activity relationship (SAR)
study on this class of compounds was conducted, showing that each of the three hydroxy groups, at C-3,
C-5, and C-6, is required for improved anti-tumoral activities and decreased cytotoxicities.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Received 26 July 2011
Received in revised form
8 September 2011
Accepted 8 September 2011
Available online 16 September 2011
Dedicated to Prof. Gerhard Spiteller on the
occasion of his 80th birthday.
Keywords:
Triphyophyllum peltatum
Dioncoquinones A-E
Naphthoquinones
Antitumor activity
Multiple myeloma
1. Introduction
1), via a mode-F type polyketide folding [6]. The phylogenetically
‘young’ transamination step of this unique biosynthetic pathway,
Tropical lianas of the small palaeotropic families Dio-
ncophyllaceae and Ancistrocladaceae, including Triphyophyllum
peltatum Airy Shaw and Ancistrocladus abbreviatus Airy Shaw
from West Africa, are the only plants known to produce naphthyl
isoquinoline alkaloids [1], like, e.g., dioncophylline A (4) [2] (see
Scheme 1). These natural products feature unique structures,
usually with rotationally hindered and thus stereogenic biaryl
axes, and are characterized by their promising antiprotozoal
activities against Plasmodium, Leishmania, and Trypanosoma
parasites [3], and by their unprecedented biosynthetic origin
[4,5]. Feeding experiments showed that both parts of the mole-
cule, the isoquinoline and the naphthalene portions, are
biosynthetically formed from six acetate units each (see Scheme
which is required for the incorporation of nitrogen into the iso-
quinoline moiety [5], is highly susceptible to chemical, physical,
or biotic stress, then leading to the formation of naph-
thoquinones like plumbagin (1) and droserone (2), instead [7].
These naphthoquinones, which are produced as phytoalexins [8]
by several other related plant families [8,9], have been shown to
possess anti-tumoral [10] and anti-malarial [11] effects.
Our studies have revealed that a systematic change of the
procedure for the cultivation of T. peltatum cell cultures [12] can
lead to an enhanced solidification of the medium, thus prevent-
ing the calli from sinking down, thus exposing the cells to more
oxidative conditions, which, in turn, favor the formation of novel,
even more highly oxygenated naphthoquinones [5]. We have
recently reported on the isolation and structural elucidation of
nine naphthoquinones, among them dioncoquinones A (5) and B
(6) [13]. Dioncoquinone B (6) showed a high and specific activity
against Leishmania major, while no or weak activities against
other protozoic parasites were observed. Furthermore,
compound 6 strongly induced apoptosis in human tumor cells
* Corresponding author.
E-mail address: bringman@chemie.uni-wuerzburg.de (G. Bringmann).
Present address: Department of Chemistry, Ludwig-Maximilians-Universität,
1
Munich, Butenandtstr. 5-13, Haus F, D-81377 Munich, Germany
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.012

G. Bringmann et al. / European Journal of Medicinal Chemistry 46 (2011) 5778e5789
5779
O
O
O
further
oxy -
genation
HO
HO
OH
Me
biotic,
Me
physical, or
O
chemical stress
plumbagin (1)
droserone (2)
OH
OH
6 acetate /
malonate
Me
Me
Me
O
O
aldol
oxidative
biaryl
R
O
O
Me
P
condensation,
decarboxylation
O
N
R
H
coupling
OH
Me
MeO
reductive
amination
COSCoA
MeO
hexaketide
precursor 3
dioncophylline A (4),
a naphthylisoquinoline alkaloid
blocked
under stress
Me
R
N
OH
Me
Scheme 1. Biosynthesis of the naphthyl isoquinoline alkaloid dioncophylline A (4), and stress-induced formation of naphthoquinones like plumbagin (1) and droserone (2), all from
six acetate/malonate units via the postulated joint hexaketide 3.
derived from two different B cell malignancies, namely B cell
lymphoma and multiple myeloma, without any significant
toxicity toward normal peripheral mononuclear blood cells [13].
In this paper, we report on the first total synthesis of dio-
ncoquinone B (6) using three different synthetic approaches, and
on the preparation of a range of congeners of 6, which were then
investigated in a first systematic structure-activity relationship
(SAR) study for these naphthoquinone compounds. In addition,
using oxidative cultivation conditions, we were able to isolate
three further new dioncoquinone-related metabolites: dio-
ncoquinones C (7), D (8), and E (9), and a known [14] one, 8-
hydroxydroserone (10) (see Fig. 1). Moreover, our synthetic
strategy enabled us to complete the first total synthesis of the
anti-tumoral naphthoquinone 7.
2. Results and discussion
2.1. Synthetic chemistry
2.1.1. The first route to dioncoquinone B (6): by a Stobbe approach
The synthesis of highly substituted naphthoquinones has been
well studied during the past decades [15]. Many strategies have
been developed to build up these systems, among them the Dötz
reaction [16], the Hauser-Kraus annulation [17], and [4 þ 2]-cyclo-
additions [18]. In analogy to previous work [19], our initial approach
to dioncoquinone B (6) was based on the well-developed Stobbe-
condensation route to a naphthalene, which was then oxidized and
modified to provide the required naphthoquinone. This pathway,
thus, started with the synthesis of the naphthalene 15 from the
known [19] bromo veratrum aldehyde 11 (see Scheme 2). A Stobbe
condensation [20] with dimethyl succinate resulted in the forma-
tion of the E-configured (NMR) acid 12, which was cyclized in acetic
anhydride in the presence of sodium acetate [21] to produce the
naphthalene 13.2 Using palladium on charcoal in methanol in the
presence of K2CO3 [22], the bromine and the acetate protecting
O
O
OH
OH
OH
Me
OR
Me
HO
RO
MeO
O
O
5: R = D-Glu
6: R = H
7: R = H
8: R = Me
O
O
OH
OH
MeO
HO
OH
OH
Me
Me
8
O
O
OH
10
2
Initial attempts to cyclize the corresponding non-brominated analog of 12 had
9
resulted in an exclusive formation of the bromine-free analog of the undesired
regioisomer of 13, showing the necessity to block the para-cyclization reaction by
a bromine substituent.
Fig. 1. Higher-oxygenated naphthoquinones isolated from T. peltatum cell cultures,
dioncoquinones A (5), B (6), C (7), D (8), and E (9), and 8-hydroxydroserone (10).

5780
G. Bringmann et al. / European Journal of Medicinal Chemistry 46 (2011) 5778e5789
MeO
Br
O
MeO
MeO
1
OH
KOtBu, MeOH
1
COOH
X
MeO
MeO
1. iPrCyNH, n-BuLi
DMS, 90%
Et N
2
H
2.
MeO
2
COOMe
Me
Me
X
Me
1
X
Br
12
O
2
15: X = OMe, X = H,
19
20
11
31%
1
2
2
Br
21: X = OMe, X = OMe,
X
21%
NaOAc, Ac O, H2O
2
140 °C, 60%
1
2
22: X = H, X = H,
35%
H , Pd/C
MeO
MeO
MeO
OH
2
MeO
OAc
Scheme 3. Synthesis of naphthols via a DielseAlder route.
K CO ,
2
3
MeO
MeO
MeO
HO
MeO
MeOH, 95%
COOMe
COOMe
a natural product that we had previously isolated from the related
plant, A. abbreviatus [13]. O-demethylation of 18 with boron tri-
bromide furnished dioncoquinone B (6) in a 98% yield, thus
completing its first total synthesis (see Scheme 2).
This first synthetic route, although reliably providing access to
dioncoquinone B (6) and offering the possibility to synthesize
derivatives of this natural product by varying the starting materials,
contains some tedious isolation and purification steps, which
prompted us to explore less time-consuming alternatives. The
improved second-generation synthesis of the same naphthalene
intermediate 15 is discussed in the following section.
Br
13
14
1. LiAlH , Et O,
4
2
96%
2. Pd/C, H , HCl
2
MeOH, 98%
OH
O
MeO
O
MeO
Me
Me
16
15
2.1.2. DielseAlder route to precursors of dioncoquinone B (6) and
dioncoquinone C (7), and to the related naphthol 22
For a better access to naphthols 15, 21, and 22, we envisioned
a strategy based on the DielseAlder (DA) reaction between
CuCl, air
MeCN, 66%
1. H O ,
2
2
O
O
O
O
O
O
MeO
K CO ,
MeO
MeO
2
3
OH
Me
1. SOCl ,
2
THF,
MeO
MeO
X
MeO
X
reflux
97%
OH
NEt
2
2. HNEt
2
2. conc. H SO ,
2
4
Me
silica gel,
THF, 95%
23, 24
25 (99%), 26 (80%)
18
17
sec-BuLi, TMEDA,
BBr
3
,
°C,
THF 90
CH Cl , rt, 98%
2
2
.
MgBr 2Et O
2
2
O
Br
OH
O
O
OH
Me
MeO
MeO
MeO
X
MeO
X
Me
NEt
NEt
2
2
29
MgBr
O
6
Me
Scheme 2. First total synthesis of dioncoquinone B (6), via a Stobbe-condensation route.
30 (60%), 31 (45%)
27, 28
MeLi, 78 °C
0 °C
groups were removed to give the free naphthol 14 in an almost
quantitative yield. Reduction of the ester group using LiAlH4 [22]
and palladium on charcoal [22] provided the methylnaphthalene
15 [23] in a 94% yield over two steps. By oxidation of 15 with CuCl
in the presence of air [24], the para-naphthoquinone 17 [23] was
obtained in a 66% yield, accompanied by the respective ortho-
MeO
OH
X
MeO
X
15, 23, 25, 27, 30 H
16, 24, 26, 28, 31 OMe
Me
quinone 16 (ratio 6.5:1). The required
target molecule was introduced by epoxidation of the 2,3-double
bond [25] of 17 followed by ring opening [26] of the resulting
-epoxydiketone. Under optimized conditions using sulfuric acid on
silica gel, this reaction sequence provided ancistroquinone C (18),
a-hydroxy functionality of the
15 (99%), 21 (80%)
a,
b
Scheme 4. Synthesis of the key intermediates 15 and 21 by Grignard reaction of the
benzamides 25 and 26, and 2-methylallyl bromide (29), and MeLi-induced cyclization.

G. Bringmann et al. / European Journal of Medicinal Chemistry 46 (2011) 5778e5789
5781
Table 1
benzynes as dienophiles and lithiated unsaturated amides as
Yields of 7, 35, and 36 using different reaction conditions in the last step.
dienes established by Watanabe et al. [23]. This required the
amide 19 and 3,4-dimethoxybenzyl bromide (20) as starting
materials for the DA reaction, through which 15 and its conge-
ners 21 and 22 were prepared, with different substitution
patterns in the left ring part. This synthetic route permitted
a concise access to several derivatives of dioncoquinone B (6).
The yield of the naphthalene products depended on the number
of electron-donating groups in the benzyne ring. The best results
were achieved using mono- and di-substituted bromobenzenes
(see Scheme 3).
Entry
BBr3 (eq.)
Time (h)
T (ꢁC)
7 (%)a
35 (%)a
36 (%)a
1
2
3
4
2
3
10
10
2
2
2
ꢀ78
0
41
67
0
0
10
13
98
0
15
15
0
ꢀ78/0
ꢀ78/0
ꢀ78/rt
16
a
Yields after normal-phase column chromatography and preparative HPLC on
reversed-phase material.
at ꢀ78 ꢁC [27], 30 and 31 were converted to the desired naph-
thalenes 15 and 21 in excellent yields (see Scheme 4). This route
enabled us to synthesize these key intermediates in a very short
manner and with high yields.
With this DielseAlder route to the intermediates 15, 21, and 22,
a significantly shorter synthetic route to dioncoquinone derivatives
was established, yet in unsatisfactory yields.
2.1.3. The Grignard approach to precursors of dioncoquinone B (6)
and dioncoquinone C (7)
2.1.4. Synthesis of dioncoquinone C (7) and its analogs
This good synthetic access to several differently substituted
naphthalene compounds 15 and 21 now permitted to synthesize
a small library of further dioncoquinone B analogs, among them the
natural product dioncoquinone C (7) (see Scheme 5 and Table 1).
Therefore, another pathway to naphthols 15 and 21 was
established, this time based on the addition of a Grignard reagent
derived from benzamides 25 and 26 to an allyl bromide [27]. This
route started with the commercially available benzoic acids 23 and
24, which were converted to the known [28,29] benzamides 25
and 26 using procedures described for other compounds [30].
After the directed ortho-deprotonation of 25 and 26 using sec-BuLi,
the resulting lithium species was transmetallated with
MgBr2∙2Et2O [31], providing Grignard reagents 27 and 28,
respectively. Their reaction with 2-methylallyl bromide (29)
resulted in the formation of the new ortho-allyl benzamides 30
and 31 in satisfactory yields. By treatment with methyl lithium
2.1.5. Synthesis of the dioncoquinone B analog 39
For SAR studies on dioncoquinone compounds, derivatives of
dioncoquinone B (6) with a different OH and OMe substitution
pattern were prepared. To synthesize 6,7-substituted analogs,
which were difficult to access by the described methods, we started
with the known naphthol 37 [32], which was oxidized to the
quinone 38 [33]. Introduction of a hydroxy group at C-3 over three
steps furnished the new analog 39 of 6 (see Scheme 6).
MeO
OH
2.1.6. Synthesis of the dioncoquinone B analog 43
MeO
MeO
To evaluate the contribution of the 5-OH function to the anti-
MM activity of dioncoquinone B (6), compound 43, i.e., the analog
without that hydroxy group, was prepared. The synthesis started
with the preparation of the new triflate 41 by treatment of the
known [22,34] naphthol 40 with triflic anhydride (see Scheme 7).
Selective oxidation of 41 using periodic acid and chromium trioxide
[35] at 0 ꢁC afforded the new naphthoquinone 42. Epoxidation of
the 2,3-double bond in 42 with concomitant cleavage of the triflate
ester, followed by epoxide opening, produced the dihydroxynaph-
thoquinone 43. For the preparation of the monophenolic derivative
Me
21
CuCl, O , MeCN
2
O
O
O
MeO
MeO
MeO
O
MeO
MeO
O
OH
MeO
Me
Me
CuCl, O
2
MeO
MeO
MeO
MeO
MeCN, 83%
32 (45%)
33 (40%)
Me
Me
O
1. H O , 2 N NaOH,
2
2
2
37
H O/MeOH, 99%
H O , K CO ,
38
1.
2.
2
2
2
3
conc. H SO ,
2.
2
4
THF, 99%
conc. H SO ,
silica gel, THF, 99%
2
4
silica gel, THF, 99%
O
O
1
3. BBr , CH Cl ,
MeO
R O
3
2
2
BBr ,
CH Cl ,
OH
Me
OH
Me
rt, 98%
3
2
5
4
2
O
R O
6
3
MeO
MeO
2
OH
5
8
4
2
6
7
3
HO
HO
3
7
R O
1
O
O
2
Me
1
R1 R2 R3
O
34
7
H
H
H
H
Me
H
39
35
36
H
Scheme 6. Synthesis of the 5-deoxy-7-hydroxy derivative 39 of dioncoquinone B (6),
i.e., with a hydroxy pattern shifted to C-6 and C-7. For reasons of comparability with
the ‘parent compound’ dioncoquinone B (6), the framework numbering was adapted to
that of 6 for all naphthoquinones prepared in this manuscript.
H
Me
Scheme 5. Synthesis of derivatives of dioncoquinone B (6) possessing an additional
oxygen function at C-7.

5782
G. Bringmann et al. / European Journal of Medicinal Chemistry 46 (2011) 5778e5789
8-hydroxydroserone (10, see Fig. 1) were isolated from this
productive plant species.
HO
TfO
Tf O, NaH,
2
CH Cl , 98%
The calli of the cell cultures were removed from the medium,
lyophilized, ground, and extracted with CH2Cl2/MeOH (v/v 1:1).
The crude extract was subjected to an ion-exchange column to
remove naphthyl isoquinoline alkaloids, followed by normal-phase
chromatography permitting isolation of three main naph-
thoquinones: droserone (2) and dioncoquinones A (5) and B (6)
[13]. In the course of these investigations, three further naph-
thoquinones, 7ꢀ9, were found in trace quantities, but easily
recognized already by their UV absorption spectra. Specifically, the
maximum UV absorption, around 419 nm, is characteristic of 3,5-
dihydroxy substituted naphthoquinones [38]. In addition, a 3,5,8-
trihydroxy substituted naphthoquinone, 10, was found, possessing
a maximum absorption at 485 nm. All these natural products were
isolated and purified by preparative HPLC and characterized by
1H-, 13C- and 2D-NMR spectroscopy.
2
2
Me
Me
Me
Me
40
41
H IO , CrO ,
5
6
3
MeCN, 0 °C,
35%
O
O
O
O
H O ,
1.
2
2
K CO ,
OH
Me
2
3
HO
TfO
THF, 92%
3
2
2. conc. H SO ,
2
4
silica gel, THF,
98%
42
43
Naphthoquinones 7ꢀ9 all showed one aromatic proton by 1H
NMR, suggesting that their ring systems were higher-substituted as
compared to those of dioncoquinones A (5) and B (6). The highly
polar dioncoquinones C (7) and E (9) were found to have, both,
molecular formulas of C12H10O6, as deduced from the number of
signals in the 13C NMR spectrum and from HRESIMS, which is more
than that of 6 by 30 units (corresponding to CH2O). The substitution
pattern of these naphthoquinones was established using 2D-NMR
spectroscopy. The spectra of dioncoquinone C (7) showed HMBC
Et NOH,
dioxane,
4
H O ,
1.
2.
2
2
50%
K CO ,
2
3
THF, 92%
(Me) SO ,
O
O
O
O
2
4
OH
K CO ,
HO
O
Me
2
3
acetone, 96%
Me conc. H SO ,
3.
2
4
silica gel,
THF, 96%
correlations from the methyl protons at
d
1.98 (2-CH3) and the only
184.2 (C-
44
45
aromatic proton at 7.27 (H-8) to the carboxyl carbon at
d
d
Scheme 7. Synthesis of dioncoquinone
at C-5.
B derivatives without a hydroxy function
1), suggesting that this aromatic proton was located at C-8 (see
Fig. 2). The NOESY correlation between H-8 and the methoxy group
at d 4.01 indicated that the latter was at C-7. In the naphthoquinone
9, by contrast, no such NOESY correlation was observed between
the methoxy group and the aromatic proton, while the methoxy
45 [36], the naphthol 44 obtained from the triflate 42 [37] was
epoxidized, O-methylated, and then converted to the target
compound 45 under acidic conditions.
protons at
d
3.91 (6-OCH3) and H-8 at
d 7.09 displayed HMBC
correlations to the carbon signal at
d
139.7 (C-6), indicating that the
methoxy group was located at C-6 in 9. In the structure of naph-
thoquinone 8, an additional O-methyl group was found to be linked
to the oxygen function at C-3 as compared to that of 7. The fourth
naphthoquinone isolated from the callus cultures was identified as
the known 8-hydroxydroserone (10), previously found in Droser-
aceae and Nepenthaceae [14], but not yet in T. peltatum. Its struc-
ture has one more chelated hydroxy group as compared to
compounds 7ꢀ9 (see Fig. 1).
2.2. New natural naphthoquinones by isolation work
In addition to the above described synthetic work, the isolation
of similar naphthoquinones from cell cultures of T. peltatum Airy
Shaw [13] proved to be a rewarding source of further related
analogs, too. Thus, three new natural products, dioncoquinones C
(7), D (8), and E (9, see Fig. 2), and the known [14] naphthoquinone
From a biosynthetic point of view, the new naphthoquinones
7ꢀ9 and the known one, 10, are apparently closely related to their
less oxygenated analogs, plumbagin (1) and droserone (2, see
Scheme 1).
O
HO
OH
HO
4.01
2
1
3. SAR studies
7
MeO
Me
8
1.98
O
H
The promising results of the biological screening of dio-
ncoquinones A (5) and B (6) [13], especially the high and selective
activity of 6 against multiple myeloma (MM) cells, prompted us to
184.2
7
7.27
3.91
O
O
HO
OH
6
MeO
HO
139.7
Table 2
EC50 values (
mM) of INA-6 multiple myeloma cells and peripheral mononuclear
Me
blood cells (PBMCs) treated with compounds 1, 2, or 5ꢀ9, or with melphalan.
8
1.96
H
1
2
5
6
7
8
9
Melphalan
7.09
INAe6a
PBMCs
0.8
0.8
>100
NR
29
NR
11
NR
14
NR
80
NR
100
NR
2 [13]
3 [13]
185.9
9
Fig. 2. Selected NMR data of 7 (in CD3COCD3) and 9 (in CD3OD): 1H and 13C NMR shifts
in ppm), NOESY correlations (red arrow) and HMBC couplings (blue arrows) indic-
ative for their constitutions. (For interpretation of the references to color in this figure
NR: not reached.
a
(
d
Multiple myeloma cells were treated with different concentrations of 1, 2, or
5ꢀ9, or melphalan. The viable fractions of the treated cells were determined by
annexin V-FITC/PI staining.
legend, the reader is referred to the web version of this article.)

G. Bringmann et al. / European Journal of Medicinal Chemistry 46 (2011) 5778e5789
5783
Table 3
necessary for the anti-MM activities of these compounds, too. It is
worth mentioning that shifting the OH groups along the aromatic
ring has a significant impact on the biological activities of naph-
thoquinones. Hydroxy shifts from position 5, 6e6, 7 (structure 39)
or 5, 8 (structure 10) both result in a decrease of the activities of the
corresponding compounds. The introduction of an additional OH
function in position 7 of 6 as in the naphthoquinone 35 does not
have any significant effect on the anti-MM activity of this
compound, while its toxicity is increased. No pronounced toxicity
was observed, however, in the 7-O-methylated derivative of 35,
dioncoquinone C (7), while its bioactivity was still high (Table 2).
Among the numerous derivatives tested, 7 was the most active
candidate, with no measurable cytotoxicity against peripheral
mononuclear blood cells (PBMCs). This activity is highly specific,
and thus rewarding, because in our tests all other mixed OH/OMe-
or completely OMe-substituted structures were entirely inactive
(structures 18, 34, 45, and 47ꢀ50).
EC50 values of INA-6 multiple myeloma cells and PBMCs treated with
naphthoquinones.
Compd.
X1
X2
X3
X4
X5
EC50
(
m
M)
EC50 (mM)
(INA-6)a
(PBMC)b
10
17
18
32
34
35
38
39
43
44
45
46
47
48
49
50
OH
H
OH
H
OH
OH
H
OH
OH
H
OH
H
OH
OMe
OH
H
OH
OMe
OMe
OMe
OMe
OH
H
H
H
H
H
H
H
H
H
OMe
OMe
OH
OMe
OH
H
H
H
H
H
H
OMe
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>100
15
>100
8
>100
7
6
52
>100
13
>100
75
>100
>100
>100
4
NR
13
NR
7.5
NR
70
6.5
NR
NR
17
OMe
OMe
OMe
OMe
OH
OMe
OH
OH
OH
OMe
OH
OMe
OH
OMe
H
In summary, for a good balance of activity versus toxicity, it
seems essential to have OH groups in positions 3, 5, and 6, while
position 7 has little influence, which can be seen from the similar
activities of dioncoquinone B (6), dioncoquinone C (7), and 7-
hydroxydioncoquinone B (35) (Table 2 and Table 3). The role of
an OH group in position 8 is not quite clear, but e at least in
combination with the lack of oxygen functions at C-6 and C-7 e it
seems to lower the activity as seen in structure 10.
50
OH
OH
OH
H
100
NR
NR
NR
16
OMe
NR: not reached.
a
Activity against the multiple myeloma cell line INA-6.
Cytotoxicity against normal peripheral mononuclear blood cells (PBMC).
b
4. Conclusion
test the biological activities of the newly synthesized naph-
thoquinones, of their synthetic intermediates, and of the isolated
natural analogs. Dioncoquinone C (7) showed a similar activity
against the MM cells as that of dioncoquinone B (6), but it was
inactive against peripheral mononuclear blood cells (PBMCs). Some
of the compounds induced strong apoptosis in MM cell lines in
concentrations comparable to that of the melphalan, which is a well
known DNA-alkylating agent [39] routinely used in a standard
therapy against multiple myeloma [40]. Dioncoquinones D (8) and
E (9) were less active than 6 and 7 (see Table 2).
For a first synthetic access to the anti-tumoral product dio-
ncoquinone B (6) in sufficient quantities for in vivo tests, three
approaches are described in this paper. The third strategy, based on
a directed ortho metalation (DOM) reaction, proved to be the best
one as compared to the first two approaches, which required either
too many steps or gave too low overall yields. Utilizing the third
strategy, the likewise highly anti-tumoral metabolite dio-
ncoquinone C (7) was synthesized, too. For the elaboration of the
first preliminary SAR studies, a number of dioncoquinone B analogs
were obtained by synthesis and by isolation from cell cultures of T.
peltatum. Among them, only the new dioncoquinone C (7) strongly
induced apoptosis in the multiple myeloma cell line INA-6 without
any significant toxicity. Thus, the two natural products dio-
ncoquinones B (6) and C (7) constitute two promising basic struc-
tures to develop anti-MM candidates. Remarkably both anti-
tumoral compounds have three hydroxy functions at C-3, C-5, and
C-6, which are required for their biological properties. Extended
SAR studies to further optimize the structures and to explore the
in vivo anti-MM activity of 6 and 7 and their analogs are currently in
progress.
To identify the pharmacophore of these molecules, several
synthetic intermediates and isolated compounds were examined
for their biological activity against multiple myeloma cells. The
results are summarized in Table 3.
The first results of our biological tests enabled us to localize the
preliminary structural elements of naphthoquinone derivatives
that are responsible for their anti-MM activity and cytotoxicity. To
examine the individual roles of the hydroxy functions at the C-3,
C-5, and C-6 positions in dioncoquinone B (6), we synthesized or
isolated derivatives that each contain only two hydroxy groups. The
test results (see structure 2 in Table 2 and structures 43 and 46 in
Table 3) show that removal of one OH group in any of these posi-
tions leads to an almost complete loss of bioactivity. The observa-
tion that substance 46 bearing two hydroxy groups at C-5 and C-6
exhibits lower activity and higher toxicity than 6, combined with
the activities of compounds 43 and 44, jointly evidence that the
lack of a hydroxy function at C-3 makes the compound more toxic
against normal blood cells. This tendency is supported by the
pairwise comparison of the properties of compounds 17 and 18, of
32 and 34, and of 38 and 49 with respect to the structural variation
at C-3 (see Table 3). A possible reason for this is that the hydroxy
group and other substituents at C-3 may block Michael addition
reactions of possible nucleophiles (e.g., SH groups of proteins) in
5. Experimental
5.1. Instrumentations and chemicals
Melting points were determined with a Kofler hot stage micro-
scope (Reichert) and are uncorrected. IR spectra were measured with
a Jasco FT/IR-410 spectrometer and are reported in wave numbers
(cmꢀ1). NMR spectra were recorded either on a Bruker AC 250,
a Bruker AV 400 or a Bruker DMX 600 spectrometers at ambient
temperature. The chemical shifts are given in
d units (ppm) taking
the signals of the deuterated solvents as internal reference for 1H and
13C NMR spectroscopy; the coupling constants J are given in Hertz
(Hz). Mass spectra were recorded on a Finnigan MAT 8200 mass
spectrometer at 70 eV for EI and on a Bruker Daltonics micrOTOF-
focus for ESI. All reactions with air and/or moisture sensitive mate-
rial were carried out in flame dried glasware using the Schlenk tube
the cell to the
a,b-unsaturated carbonyl fragments in naph-
thoquinones 17, 32, and 38, thereby inhibiting a variety of cellular
functions that direct the cells into apopotosis [41]. On the other
hand, the possibility of a Michael addition reaction seems to be

5784
G. Bringmann et al. / European Journal of Medicinal Chemistry 46 (2011) 5778e5789
technique under an inert nitrogen or argon atmosphere. Preparative
HPLC was performed on a SymmetryPrep-C18 column (Waters;
19 ꢂ 300 mm); flow rate of 10 mL/min; mobile phase: (A) CH3CN
(0.05% trifluoroacetic acid), (B) H2O (0.05% trifluoroacetic acid);
room temperature; detection by a photodiode array.
9.63 (s,1H, OH).13C NMR (CDCl3)
d 52.4, 56.9, 63.3,109.5,115.6,120.6,
122.4, 127.1, 127.2, 130.4, 142.9, 149.3, 153.7, 167.3. EI-MS (70 eV) m/z
(%): 262 (100), 247 (48), 231 (9), 219 (15). Anal. calcd for C14H14O5: C
64.12; H 5.38; found: C 63.76; H 4.97.
5.2.1.4. 4-Hydroxy-5,6-dimethoxy-2-hydroxymethylnaphthalene. The
5.2. Chemistry
ester 14 (139 mg, 530 mmol) in dry Et2O (3 mL) was added dropwise
to a stirred suspension of lithium aluminum hydride (61.0 mg,
1.59 mmol) in 30 mL Et2O under a nitrogen atmosphere at room
temperature. The solution was stirred for 4 h, then water and half-
concentrated aqueous HCl solution (20 mL) were added. The crude
mixture was extracted with CH2Cl2 (3 ꢂ 20 mL), and the organic
layer was dried over Na2SO4, concentrated in vacuo and the residue
was purified by crystallization in CH2Cl2 to afford the 2-
5.2.1. The first route to dioncoquinone B (6)
5.2.1.1. (E)-4-(20-bromo-40,50-dimethoxyphenyl)-3-methoxycarbonyl-
3-butenoic acid (12). Dimethyl succinate (775 mg, 5.30 mmol) and 2-
bromo-4,5-dimethoxybenzaldehyde [19] (11) (500 mg, 2.04 mmol)
were added to a solution of potassium-tert-butoxide (916 mg,
8.16 mmol)in dry methanol(8 mL). The mixturewasrefluxed for 48 h,
then poured into stirred aqueous 10% HCl solution at 0 ꢁC, and
extracted with EtOAc (3 ꢂ 80 mL). The extract was washed with water
(100 mL) and extracted with 0.5 N aqueous NaOH (3 ꢂ 50 mL). The
aqueous phase was acidified with half-concentrated HCl solution, and
extracted with EtOAc (3 ꢂ100mL). Theorganiclayerwaswashedwith
H2O, saturated aqueous NaCl, dried over Na2SO4, and concentrated.
TheresiduewasrecrystallizedfromEtOActoafford12 asa yellowsolid
hydroxymethylnaphthalene as a yellow solid (119.0 mg, 510
m
mol,
96%). Mp 80ꢀ83 ꢁC (CH2Cl2). IR (KBr, cmꢀ1
) n 3234, 2937, 2931, 2362,
1643, 1613, 1578, 1514, 1489, 1367, 1148, 1044, 949, 833, 646. 1H NMR
(CDCl3) d 4.03 (s, 3H, OCH3), 4.13 (s, 3H, OCH3), 4.70 (s, 2H, CH2), 5.66
(s, 1H, OH), 6.82 (d, J ¼ 1.4 Hz, 1H, AreH), 7.30 (s, 1H, AreH), 7.43 (d,
J ¼ 9.0 Hz, 1H, AreH), 7.64 (d, J ¼ 9.0 Hz, 1H. AreH), 9.55 (s, 1H, OH).
13C NMR (CDCl3)
d 57.1, 62.2, 65.5, 109.4, 115.7, 116.6, 117.8, 125.3,
(660 mg, 1.84 mmol, 90%). Mp 148ꢀ150 ꢁC (EtOAc). IR (KBr, cmꢀ1
)
n
131.6, 138.5, 143.2, 147.5, 153.9. EI-MS (70 eV) m/z (%): 234 (100), 219
(54), 191 (10). Anal. calcd for C13H14O4: C 64.12; H 5.38; found: C
63.98; H 5.33.
2961, 2836, 2361, 2111, 1717, 1697, 1596, 1502, 1466, 1436, 1387, 1330,
1262, 1206, 1167, 1092, 1023, 920, 871, 814, 770, 745, 607. 1H NMR
(CDCl3)
(s, 3H, OCH3), 7.04 (s,1H, C]CH), 7.09 (s,1H, AreH), 7.93 (s,1H, AreH),
10.72 (br s, 1H, COOH). 13C NMR (CDCl3)
34.5, 53.1, 56.5, 56.5, 113.1,
d 3.49 (s, 2H, CH2), 3.87 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.90
5.2.1.5. 5,6-Dimethoxy-2-methyl-4-naphthol (15). 5,6-Dimethoxy-
d
4-hydroxy-2-hydroxymethylnaphthalene (100.0 mg, 43.0
mmol)
115.5,115.8,124.9,126.8,142.9,148.7,150.6,168.9,173.3. EI-MS (70 eV)
m/z (%): 358 (3), 279 (100), 220 (11). MS (ESI) exact mass calcd for
C14H16BrO6: 359.01248 [M þ H]þ; found: 359.01248 [M þ H]þ.
was stirred in a mixture of methanol, concentrated HCl (100
m
L),
and palladized charcoal (5%, 5.0 mg) for 40 min under a hydrogen
atmosphere at room temperature. After the catalyst had been
separated by filtration through Celite, the eluent was diluted with
water and half-concentrated HCl solution, and extracted with
EtOAc (3 ꢂ 10 mL). The combined organic layers were dried over
Na2SO4, and concentrated by evaporation. Crystallization of the
residue from CH2Cl2 gave 15 as a pale-yellow solid (92.0 mg,
5.2.1.2. Methyl 4-acetoxy-8-bromo-5,6-dimethoxy-2-naphthoate (13).
The acid 12 (2.67 g, 7.43 g) and anhydrous sodium acetate (612 mg,
7.46 mmol) in acetic anhydride (300 mL) were refluxed under dry
nitrogen for 2 h. After addition of H2O (1300 mL), the reaction
mixture was stirred for 2 h at room temperature until the acetic
anhydride had been hydrolyzed. The crude product was neutralized
with saturated aqueous NaHCO3 solution, and extracted with EtOAc.
The organic layer was dried over Na2SO4, concentrated in vacuo, and
chromatographed on silica gel to give product 13 (1.70 g, 4.44 mmol,
420
m
mol, 98%). Mp 36ꢀ37 ꢁC (EtOAc); Ref. [23], 35ꢀ37 ꢁC (petro-
leum ether). Spectroscopic data (IR, 1H NMR, MS) in agreement
with those reported in the literature [23].
5.2.1.6. 5,6-Dimethoxy-2-methyl-1,4-naphthoquinone (17). A suspen-
60%). Mp 110ꢀ113 ꢁC (EtOAc). IR (KBr, cmꢀ1
)
n
3097, 2956, 2849,
sion of CuCl (25.0 mg, 250
mmol) and methylnaphthol 15 (70.0 mg,
2117, 1767, 1715, 1593, 1467, 1439, 1335, 1281, 1255, 1227, 1195, 1100,
320 mol) in acetonitrile (12 mL) was stirred for 2 h with a strong
m
1046, 972, 821, 765, 729, 607. 1H NMR (CD3COCD3)
d
2.38 (s, 3H,
current of air bubbling through it. The reaction mixture was diluted
with water (10 mL) and extracted with CH2Cl2 (3 ꢂ 20 mL). The
organic layer was dried over Na2SO4, concentrated and purified by
column chromatography on silica gel (n-hexane/EtOAc) to give 17 as
CH3), 3.90 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 4.09 (s, 3H, OCH3), 7.67
(d, J ¼ 1.6 Hz, 1H, AreH), 7.97 (s, 1H, AreH), 8.78 (d, J ¼ 1.6 Hz, 1H,
AreH). 13C NMR (CD3COCD3)
d 20.8, 52.9, 57.5, 62.0, 119.9, 120.9,
121.8, 127.5, 128.8, 129.2, 143.3, 147.4, 147.4, 153.1, 166.4, 170.1. EI-MS
(70 eV) m/z (%): 382 (21), 340 (100), 287 (28). Anal. calcd for
C16H15BrO3: C 50.15; H 3.95; found: C 49.70; H 3.71.
an orange-colored solid (49.2 mg, 210
m
mol, 66%). Mp 182ꢀ185 ꢁC
(EtOAc); Ref. [23], 184ꢀ185 ꢁC (petroleum ether). Spectroscopic
data (IR, 1H NMR, MS) in agreement with those reported in the
literature [23].
5.2.1.3. Methyl 4-hydroxy-5,6-dimethoxy-2-naphthoate (14). A solu-
tion of 13 (216 mg, 560
mmol) in methanol (20 mL) was stirred with
5.2.1.7. 5,6-Dimethoxy-2-methyl-1,2-naphthoquinone (16). Further-
palladized charcoal (5%, 10.0 mg) and K2CO3 (390.0 mg, 2.82 mmol)
under a hydrogen atmosphere at room temperature. After the
catalyst had been separated by filtration through Celite, the crude
mixture was diluted with water, neutralized with half-concentrated
HCl solution, and extracted with CH2Cl2 (3 ꢂ 30 mL). The organic
layer was dried with Na2SO4, and the solvent evaporated to afford
a crude product, which was purified by crystallization in CH2Cl2 to
more, a red solid (6.0 mg, 25
m
mol, 8%) ꢀw1as isolated from the above
reaction. Mp 133 ꢁC (EtOAc). IR (KBr, cm
) n 3079, 2944, 2843, 1691,
1650, 1624, 1567, 1485, 1450, 1415, 1378, 1328, 1269, 1246, 1166, 1114,
1078,1032, 991, 960, 906, 835, 813, 772, 753, 700, 641.1HNMR (CDCl3)
d
2.14 (s, 3H, CH3), 3.92 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 6.72 (s,1H, H-
3), 7.17 (d, J ¼ 8.6 Hz,1H, AreH), 7.93 (d, J ¼ 8.6 Hz,1H, AreH).13C NMR
(CDCl3)
d 15.4, 30.6, 56.2, 61.4, 117.1, 124.1, 125.6, 128.7, 133.6, 142.5,
give 14 as a yellow solid (139.0 mg, 530
m
mol, 95%). Mp 99ꢀ100 ꢁ
C
153.9, 155.3, 178.9, 181.7. MS (ESI) exact mass calcd for C13H13O4:
(CH2Cl2). IR (KBr, cmꢀ1
)
n
3303, 2944, 1728, 1612, 1578, 1510, 1487,
233.08084 [M þ H]þ; found: 233.08080 [M þ H]þ.
1371, 1272, 1215, 1093, 1055, 996, 958, 887, 809, 780, 761, 644, 620,
606. 1H NMR (CDCl3)
d
3.94 (s, 3H, OCH3), 4.01 (s, 3H, OCH3), 4.09 (s,
5.2.1.8. 5,6-Dimethoxy-2-methyl-1,4-naphthoquinone-2,3-epoxide. Aq-
3H, OCH3), 7.30 (d, J ¼ 9.0 Hz, 1H, AreH), 7.40 (d, J ¼ 1.6 Hz, 1H,
AreH), 7.69 (d, J ¼ 9.1 Hz, 1H, AreH), 8.04 (d, J ¼ 1.4 Hz, 1H, AreH),
ueous H2O2 solution (30%, 1.5 mL) was added with stirring to
naphthoquinone 17 (35.0 mg, 150 mmol) in THF (4 mL) at ambient

G. Bringmann et al. / European Journal of Medicinal Chemistry 46 (2011) 5778e5789
5785
temperature. 1 N aqueous Na2CO3 solution was added dropwise
until the orange solution had turned colorless. Stirring was
continued at ambient temperature for 1 h. The mixture was diluted
with saturated aqueous NaCl solution and extracted with EtOAc
(3 ꢂ 15 mL). The organic layer was dried with Na2SO4, and the
solvent evaporated to dryness. The crude epoxide was crystallized
stirred for another 30 min. THF was removed by evaporation and
the residue was extracted with CHCl3. The organic layer was dried
over Na2SO4 and concentrated in vacuo. The crude product was
purified by column chromatography on silica gel and crystallized to
afford the naphthol 15. as a colorless solid (1.33 g, 6.07 mmol, 31%).
Mp (36ꢀ37 ꢁC, EtOAc) and spectroscopic data in agreement with
those described in Section 5.2.1.5 and as reported in the literature
[23] (Mp 35ꢀ37 ꢁC, petroleum ether).
from EtOAc to give a colorless solid (36.0 mg, 145
m
mol, 97%). Mp
151ꢀ153 ꢁC (EtOAc). IR (KBr, cmꢀ1
) n 2925, 2900, 1706, 1687, 1581,
1488, 1451, 1423, 1343, 1277, 1235, 1212, 1171, 1147, 1093, 1060, 1045,
1023, 984, 967, 869, 845, 809, 725, 674. 1H NMR (CDCl3)
d
1.70 (s,1H,
5.2.2.2. 5,6,7-Trimethoxy-2-methyl-4-naphthol (21). Following the
protocol described above (Section 5.2.2.1), N,N-dieth-
ylsenecioamide (19) (325 mg, 2.10 mmol) and bromide 20 (1.03 g,
4.18 mmol, X1 ¼ OMe, X2 ¼ OMe) were reacted affording 21 as
CH3), 3.81 (s, 1H, CH), 3.93 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 7.18 (d,
J ¼ 8.6 Hz,1H, AreH), 7.78 (d, J ¼ 8.6 Hz,1H, AreH). 13C NMR (CDCl3)
d
14.9, 56.5, 61.9, 62.1, 62.2, 116.3, 124.9, 125.7, 126.7, 148.5, 158.9,
191.7, 191.8. MS (ESI) exact mass calcd for C13H12O5Na: 271.05769
[M þ Na]þ; found: 271.05769 [M þ Na]þ. Anal. calcd for C13H12O5: C
62.90; H 4.87; found: C 62.58; H 4.62.
a colorless solid (108 mg, 435 m
mol, 21%). Mp 80 ꢁC (petroleum
ether/ethyl acetate); Ref. [42], 80ꢀ81 ꢁC (ethanol). Since the spec-
troscopic data were not fully given in the literature [42], they are
presented here: 1H NMR (CDCl3)
d
2.39 (s, 3H, OCH3), 3.94 (s, 6H,
OCH3), 4.13 (s, 3H, OCH3), 6.63 (s, 1H, AreH), 6.83 (s, 1H, AreH), 6.97
(s, 1H, AreH), 9.34 (s, 1H, OH). 13C NMR (CDCl3)
21.7, 55.8, 61.2,
5.2.1.9. 3-Hydroxy-5,6-dimethoxy-2-methyl-1,4-naphthoquinone (anci-
stroquinone C,18). To a solution of the epoxide (20.0 mg, 81.0
mmol) in
d
THF (20 mL) silica gel (116.0 mg) and concentrated H2SO4 (100
mL)
62.2, 102.9, 110.5, 110.9, 117.3, 132.8, 137.1, 139.0, 148.3, 152.9, 155.6.
were added. The solvent was evaporated in vaccum (200 mbar) at
70 ꢁC for 20 min on a rotary evaporator. To the resultant red solid, H2O
(10 mL) was added and the mixture was extracted with CH2Cl2. The
organic layer was extracted with 5% aqueous K2CO3 solution until it
had turned colorless. The water layer was acidified with half-
concentrated aqueous HCl solution and extracted with CH2Cl2 until
the color in the water layer was colorless again. Removal of the
organic solvent gave the crude product 18 as a red solid, which was
EI-MS (70 eV) m/z (%): 248 (100), 233 (42), 205 (19).
5.2.2.3. 5-Methoxy-2-methyl-4-naphthol (22). According to the
procedure described in Section 5.2.2.1, N,N-diethylsenecioamide
(19) (1.50 g, 9.66 mmol) and bromide 20 (2.77 mL, 21.9 mmol,
X1 ¼ H, X2 ¼ H) were reacted to yield 22 as a colorless solid
(684 mg, 3.38 mmol, 35%). Mp 89 ꢁC (petroleum ether/ethyl
acetate); Ref. [23], 89 ꢁC (n-hexane). Spectroscopic data (IR, 1H
NMR, MS) in agreement with those reported in the literature [23].
purified by crystallization from CH2Cl2 (19.0 mg, 760 mmol, 95%). Mp
218 ꢁC (CH2Cl2); Ref. [13], 217 ꢁC (H2O/MeCN). All spectroscopic data
(IR, 1H NMR, 13C NMR, MS) in agreement with those of the isolated
natural product [13].
5.2.3. The third route to naphthols 15 and 21
5.2.3.1. N,N-diethyl-2,3-dimethoxybenzamide (25). A mixture of
thionyl chloride (1.40 g, 11.8 mmol, 837 mL) and benzoic acid 23
5.2.1.10. 3,5,6-Trihydroxy-2-methyl-1,4-naphthoquinone (dioncoquinone
(429 mg, 2.36 mmol) in a 10-mL one-necked flask was refluxed at
80 ꢁC overnight. The excess of thionyl chloride was evaporated
under reduced pressure to give the acyl chloride. It was added
carefully with stirring at 0 ꢁC to diethylamine (517 mg, 7.1 mmol,
B, 6). A solution of BBr3 in CH2Cl2 (1 M, 120
dropwise to a solution of naphthoquinone 18 (10.0 mg, 40.0
dry CH2Cl2 (200
L) at 0 ꢁC. The reaction mixture was stirred at room
mmol, 0.12 mL) was added
mmol) in
m
temperature for 2 h, then diluted with water (4 mL) and extracted with
CH2Cl2 (3 ꢂ 10 mL). The organic layer was extracted with 5% aqueous
K2CO3 solution, and the water layer was acidified with half-
concentrated aqueous HCl solution, extracted with CH2Cl2
(3 ꢂ 20 mL). The organic layer was dried over Na2SO4, concentrated,
and subjected to crystallization in CHCl3 affording 6 as a red solid
732 mL) in anhydrous THF (10 mL) in a 100-mL one-necked flasks.
The reaction was continued at room temperature with stirring
overnight. The mixture was diluted with water, acidified with 0.5 N
aqueous HCl solution, and neutralized with saturated aqueous
NaHCO3 solution. The solvent was evaporated under reduced
pressure and the residue was extracted with CHCl3 (100 mL ꢂ 3),
dried with anhydrous Na2SO4, concentrated in vacuo, and subjected
to column chromatography to afford the product 25. Yellow oil
(553 mg, 2.33 mmol, 99%). Spectroscopic data (IR, 1H NMR, 13C
NMR, MS) in agreement with those reported in the literature [28].
(9.1 mg, 39
m
mol, 98%). Mp 216ꢀ217 ꢁC (CH2Cl2); Ref. [13], 218 ꢁC. All
spectroscopic data (IR, 1H NMR, 13C NMR, MS) in agreement with those
of the isolated natural product [13].
5.2.2. The second route to naphthols 15, 21, and 22
5.2.2.1. 5,6-Dimethoxy-2-methyl-4-naphthol (15). Diethylamine
(180 mmol, 18.6 mL) was added slowly to a solution of senecioyl
chloride (84.4 mmol, 9.4 mL) in 220 mL dry THF under a nitrogen
atmosphere at 0 ꢁC. The mixture was kept at room temperature for
1 h to give N,N-diethylsenecioamide (19), which was purified by
distillation under reduced pressure. N-isopropylcyclohexylamide
(LCI) was prepared by adding a 1.6 M solution of n-BuLi in n-hexane
(67.6 mmol, 42.1 mL) to N-isopropylcyclohexylamine (67.6 mmol,
11.3 mL) at 0 ꢁC with stirring and kept at room temperature for
1.5 h. The n-hexane was removed in vaccum and dry THF (50.0 mL)
was added. To the freshly prepared solution of LCI in THF (50.0 mL),
N,N-diethylsenecioamide (19) (3.00 g, 19.3 mmol) was added
at ꢀ78 ꢁC under nitrogen and the mixture was stirred for 1 h. The
bromide 20 (38.6 mmol, 5.54 mL, X1 ¼ OMe, X2 ¼ H) was injected to
the flask, which was then allowed to warm to room temperature
and stirred for 18 h. Aqueous ammonium chloride solution and 10%
aqueous HCl solution were added and the reaction mixture was
5.2.3.2. N,N-diethyl-2,3,4-trimethoxybenzamide (26). In a similar
way as described above, benzoic acid 24 (500 mg, 2.36 mmol) was
converted to 26. Yellow oil (504 mg, 1.89 mmol, 80%). In the liter-
ature [29], no spectroscopic data of 26 were reported, so they are
presented here: IR (KBr, cmꢀ1) 2972, 2937, 1610, 1458, 1433, 1315,
1265, 1220, 1002, 837, 794, 758. 1H NMR (CDCl3):
d
1.01 (t, J ¼ 7.0 Hz,
3H, CH3), 1.21 (t, J ¼ 7.0 Hz, 3H, CH3), 3.15 (q, J ¼ 7.0 Hz, 2H, CH2),
3.20 and 3.40 (broad signal, 2H, CH2), 3.77 (s, 3H, OCH3), 3.86 (s, 3H,
OCH3), 3.87 (s, 3H, OCH3), 6.64 (d, J ¼ 8.4 Hz, 1H, AreH), 6.87 (d,
J ¼ 8.4 Hz,1H, AreH). 13C NMR (CDCl3):
d 12.7 (CH3), 13.9 (CH3), 39.0
(CH2), 43.1 (CH2), 56.0 (CH3O-4), 60.9 (CH3O-2), 61.6 (CH3O-3),
107.6 (C-5), 121.6 (C-6), 124.8 (C-1), 142.0 (C-2), 149.8 (C-3), 154.2
(C-4), 168.4 (CO). EI-MS (70 eV) m/z (%): 267 (19), 195 (100).
5.2.3.3. N,N-diethyl-2,3-dimethoxy-6-(2-methylallyl)benzamide (30).
Preparation of MgBr2∙2Et2O following a literature known [31]
procedure (yet in German)

5786
G. Bringmann et al. / European Journal of Medicinal Chemistry 46 (2011) 5778e5789
To 2.43 g magnesium (100 equiv) in dry diethylether (50 mL) in
5.2.4. The route to 3,5,6,7-tetrahydroxy-2-methyl-1,4-naphtho
quinone (dioncoquinone C, 7)
a three-neck flask under argon, 1,2-dibromoethane (0.5 mL) was
added by syringe injection. After the reaction had started, the
remaining 1,2-dibromoethane (7.9 mL, total 97.4 mmol) was added
under reflux. After 30 min, the flask was cooled to room temper-
ature. The lower, brown phase, containing the MgBr2∙2Et2O solu-
tion (2.62 N) was freshly utilized for the transmetalation step.
To a stirred THF solution of sec-BuLi (34.0 mL, 47.7 mmol) and
TMEDA (7.1 mL, 47.7 mmol) the benzamide 25 (10.3 g, 43.4 mmol) in
THF (100 mL) was added at ꢀ90 ꢁC under argon by syringe injection
and stirred for 1.5 h. The solution of the lithiated species was then
warmed to ꢀ78 ꢁC over 30 min and freshly prepared MgBr2∙2Et2O
(33.0 mL, 86.8 mmol) was added. The mixture was stirred for
30 min, allowed to warm to room temperature, again cooled
to ꢀ78 ꢁC, and stirred for 1 h. The freshly distilled 3-bromo-2-
methylpropene (29) (8.7 mL, 86.8 mmol) was added and the solu-
tion was allowed to warm to room temperature, and stirred over-
night. The reaction mixture was treated with saturated aqueous
NH4Cl. Removal of THF by rotary evaporation, extraction with
CH2Cl2, drying with Na2SO4, removal of the solvent, and purification
by column chromatography gave 30 as a slightly yellow liquid. Pale-
yellow oil (7.60 g, 26.0 mmol, 60%). IR (KBr, cmꢀ1) 2977, 2937, 1627,
5.2.4.1. 5,6,7-Trimethoxy-2-methyl-1,4-naphthoquinone (32). Follo-
wing the protocol described in Section 5.2.1.6, 5,6,7-trimethoxy-2-
methyl-4-naphthol (21) (760 mg, 3.10 mmol) was converted to the
quinone 32. Yellow powder (365.0 mg, 1.40 mmol, 45%). Mp 142 ꢁC
(petroleum ether/ethyl acetate); Ref. [42], 142ꢀ143 ꢁC. Since the
spectroscopic data were not fully given in the literature [42], they
are presented in the following: IR (KBr, cmꢀ1
) n 2945, 2837, 1654,
1629, 1572, 1484, 1457, 1433, 1408, 1375, 1355, 1315, 1279, 1224,
1198, 1181, 1159, 1116, 1077, 1019, 1005, 987, 934, 911, 900, 865, 820,
811, 781, 722, 701, 650, 631. 1H NMR (CDCl3):
d
2.11 (s, 3H, CH3), 3.91
(s, 3H, OCH3), 3.95 (s, 3H, OCH3), 3.99 (s, 1H, OCH3), 6.66 (s, 1H,
AreH), 7.46 (s, 1H, AreH). 13C NMR (CDCl3)
15.8, 56.4, 61.3, 61.6,
d
106.3, 119.7, 129.5, 132.5, 137.6, 145.5, 147.9, 154.0, 157.2, 183.6, 185.1.
EI-MS (70 eV) m/z (%): 262 (100), 247 (69).
5.2.4.2. 5,6,7-Trimethoxy-2-methyl-1,2-naphthoquinone(33). Further-
more, a red solid (384.0 mg, 1.46 mmol, 47%) was isolated from the
above reaction. Mp 184 ꢁC (petroleum ether/ethyl acetate). IR (KBr,
cmꢀ1
)
n
3092, 2945, 2920, 2847, 2359, 1661, 1648, 1632, 1575, 1491,
1455, 1406, 1368, 1329, 1267, 1200, 1147, 1084, 1030, 1000, 976, 964,
934, 919, 865, 809, 780, 738, 686, 668, 647. 1H NMR (CDCl3):
2.14 (s,
1430,1376,1272,1058, 890, 804.1H NMR (CDCl3):
d
1.08 (t, J ¼ 7.0 Hz,
d
3H, CH3),1.27 (t, J ¼ 7.0 Hz, 3H, CH3),1.72 (s, 3H, CH3), 3.18 (qd, J ¼ 7.0,
1.6 Hz, 2H, CH2), 3.25 (d, J ¼ 6.4 Hz, 2H, CH2), 3.61 (qd, J ¼ 7.0, 6.4 Hz,
1H, CH2), 3.85 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.62 (s, 1H), 4.85 (s,
1H), 6.88 (s, J ¼ 8.0 Hz, 1H, AreH), 6.92 (s, J ¼ 8.0 Hz, 1H, AreH). 13C
3H, CH3), 3.92 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 6.72 (s, 1H, H-3), 7.17
(d, J ¼ 8.6 Hz, 1H, AreH), 7.93 (d, J ¼ 8.6 Hz, 1H, AreH). 13C NMR
(CDCl3)
d 15.5, 56.4, 61.4, 61.5, 109.0, 118.1, 133.2, 135.6, 141.6, 143.7,
158.4, 159.1, 176.8, 181.8. MS (ESI) exact mass calcd for C14H14O5Na:
NMR (CDCl3):
d
12.5,13.5, 22.1, 38.2, 40.0, 42.7, 55.5, 61.1,112.0,112.4,
285.07334 [M þ Na]þ; found: 285.07330 [M þ Na]þ.
125.1, 128.3, 132.4, 143.8, 144.5, 150.6, 167.2. EI-MS (70 eV) m/z (%):
291 (8), 219 (100), 218 (58). MS (ESI) exact mass calcd for
C17H25NO3Na: 314.17321 [M þ Na]þ; found: 314.17350 [M þ Na]þ.
5.2.4.3. 5,6,7-Trimethoxy-2-methyl-1,4-naphthoquinone-2,3-epoxide.
According to the protocol described in Section 5.2.1.8, the naph-
thoquinone 32 (68.1 mg, 260
sponding epoxide. Pale-yellow powder (72.0 mg, 257
110 ꢁC (MeOH/H2O). IR (KBr, cmꢀ1
2979, 2946, 1681, 1654, 1631,
mmol) was converted to the corre-
5.2.3.4. N,N-diethyl-2,3,4-trimethoxy-6-(2-methylallyl)benzamide(31).
In a similar way as described above, the benzamide 26 (2.49 g,
9.33 mmol) was converted to the 2-methylallyl product 31. Pale-
yellow oil (1.35 g, 4.2 mmol, 45%). IR (KBr, cmꢀ1) 2977, 2937, 1625,
1456, 1425, 1400, 1319, 1284, 1141, 1105, 1033, 890. 1H NMR (CDCl3):
mmol, 99%). Mp
)
n
1571, 1485, 1461, 1409, 1346, 1315, 1279, 1193, 1161, 1116, 1099, 1072,
999, 987, 928, 902, 871, 820, 806, 785, 762, 731. 1H NMR (CDCl3):
d
1.70 (s, 3H, CH3), 3.79 (s, 1H, H-3), 3.94 (s, 6H, OCH3), 3.97 (s, 3H,
d
1.03 (t, J ¼ 7.0 Hz, 3H, CH3), 1.22 (t, J ¼ 7.0 Hz, 3H, CH3), 1.68 (s, 3H,
OCH3), 7.29 (s, 1 H, AreH). 13C NMR (CDCl3)
d
14.6, 56.4, 61.2, 61.6,
CH3), 3.09 (qd, J ¼ 7.0, 1.6 Hz, 2H, CH2), 3.15 (s, 2H, CH2), 3.54 (qd,
J ¼ 7.0, 6.4 Hz, 1H, CH2), 3.82 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.87 (s,
3H, OCH3), 4.70 (s, 1H), 4.84 (s, 1H), 6.52 (s, 1H, AreH). 13C NMR
62.1, 106.3,120.1, 129.0,137.6, 148.2,153.5, 157.7, 183.6, 185.1. MS (ESI)
exact mass calcd for C14H14NaO6: 301.06826 [M þ Na]þ; found:
301.06821 [M þ Na]þ.
(CDCl3):
d 12.8, 13.9, 22.5, 38.6, 41.0, 43.1, 56.2, 61.1, 61.8, 108.7, 113.1,
125.1, 131.7, 140.4, 144.0, 149.6, 153.5, 167.7. EI-MS (70 eV) m/z (%): 321
(10), 249 (100). MS (ESI) exact mass calcd for C18H27NO4Na: 344.18378
[M þ Na]þ; found: 344.18330 [M þ Na]þ.
5.2.4.4. 3-Hydroxy-5,6,7-trimethoxy-2-methyl-1,4-naphthoquinone
(34). Following the protocol described in Section 5.2.1.9, 5,6,7-
trimethoxy-2-methyl-1,4-naphthoquinone-2,3-epoxide (53.9 mg,
194
(53.0 mg, 192
m
mol) was converted to the naphthoquinone 34. Yellow powder
5.2.3.5. 5,6-Dimethoxy-2-methyl-4-naphthol (15). To the ortho-allyl
benzamide 30 (2.41 g, 8.2 mmol) in 50 mL THF, 2.2 equiv of MeLi
were added with stirring at ꢀ78 ꢁC. The reaction mixture was
slowly warmed up to 0 ꢁC over 5 h, followed by addition of satu-
rated aqueous NH4Cl solution, evaporation of the solvent, and
extraction with EtOAc. The organic layer was dried with Na2SO4,
and concentrated in vacuo. The crude product was purified by
column chromatography to afford the naphthol 15 as a colorless
solid (1.76 g, 8.1 mmol, 99%). Mp (36ꢀ37 ꢁC, EtOAc) and spectro-
scopic data in agreement with those described in Section 5.2.1.5
and reported in the literature [23] (Mp 35ꢀ37 ꢁC, petroleum ether).
m
mol, 99%). Mp 177 ꢁC (CHCl3). IR (KBr, cmꢀ1
) n
3329,
2948, 2841, 1655,1641, 1573,1486, 1460,1412,1382,1346,1258,1210,
1181, 1144, 1105, 1080, 1021, 998, 965, 910, 869, 816, 787, 761, 746,
720, 704. 1H NMR (CDCl3):
d
2.05 (s, 3H, CH3), 3.94 (s, 3H, OCH3), 3.96
(s, 3H, OCH3), 4.02 (s, 3H, OCH3), 7.54 (s, 1H, OH), 7.66 (s, 1H, AreH).
13C NMR (CDCl3)
8.5, 56.5, 61.3, 61.6, 106.8, 116.3, 117.7, 130.7, 146.6,
d
153.7, 154.7, 158.5, 178.9, 184.3. MS (ESI) exact mass calcd for
C14H14NaO6: 301.06826 [M þ Na]þ; found: 301.06821 [M þ Na]þ.
5.2.4.5. 3,5,6-Trihydroxy-7-methoxy-2-methyl-1,4-naphthoquinone
(dioncoquinone C, 7). O-Demethylation to dioncoquinone C (7) and
its two analogs 35 and 36 from the quinone 34 was achieved
following the procedure described for the preparation of dio-
ncoquinone B (6) from 18 in 5.2.1.9, with modifications of the
reaction time, the temperature, and the equivalents of BBr3 used
(see Table 1). Under different reaction conditions, with increasing
temperature from ꢀ78 ꢁC to 0 ꢁC within 2 h and 10 equiv of BBr3, 34
was converted to its 5,6-O-didemethyl derivative, dioncoquinone C
5.2.3.6. 5,6,7-Trimethoxy-2-methyl-4-naphthol (21). In
a similar
way as described above (Section 5.2.3.5), the amide 31 (3.48 g,
10.7 mmol) was converted to 21 (2.16 g, 8.6 mmol, 80%). Mp 80 ꢁC
(petroleum ether/ethyl acetate); Ref. [42], 80ꢀ81 ꢁC (ethanol). All
spectroscopic data in agreement with those described in Section
5.2.2.2.

G. Bringmann et al. / European Journal of Medicinal Chemistry 46 (2011) 5778e5789
5787
(7), with a satisfactory yield. Red powdꢀe1r (14.0 mg, 56
m
mol, 67%).
126.9,154.3,154.5,191.0,191.1. EI-MS (70 eV) m/z (%): 248 (100), 233
(17), 220 (12), 206 (24), 191 (16). Anal. calcd for C13H12O5: C 62.90;
H 4.87; found: C 61.91; H 5.12.
Mp > 340 ꢁC (ethyl acetate). IR (KBr, cm
) n 3384, 3921, 1600, 1571,
1463, 1319, 1238, 1151, 1072, 997, 804, 740. 1H NMR (CD3COCD3):
1.98 (s, 3H, 2-CH3), 4.01 (s, 1H, 7-OCH3), 7.27 (s, 1H, 8-H). 13C NMR
(CD3COCD3) 8.6 (CH3-2), 56.8 (CH3O-7), 104.8 (C-8), 109.8 (C-10),
121.0 (C-2), 125.3 (C-9), 139.4 (C-6), 150.5 (C-5), 153.6 (C-7), 154.8
(C-3), 184.2 (C-1), 185.1 (C-4). 1H NMR (CD3OD):
1.97 (s, 3H, 2-
CH3), 3.99 (s, 1H, 7-OCH3), 7.29 (s, 1H, 8-H). 13C NMR (CD3OD)
8.6 (CH3-2), 56.9 (CH3O-7), 105.1 (C-8), 110.3 (C-10), 121.5 (C-2),
d
d
5.2.5.3. 3-Hydroxy-6,7-dimethoxy-2-methyl-1,4-naphthoquinone. Acc-
ording to the protocol described in Section 5.2.1.9, 6,7-dimethoxy-2-
d
methyl-1,4-naphthoquinone-2,3-epoxide (50.0 mg, 200
mmol) was
converted to the corresponding naphthoquinone. Yellow powder
d
(28.0 mg, 110 m
mol, 56%). Mp 230 ꢁC (n-hexane/ethyl acetate);
125.5 (C-9), 140.0 (C-6), 151.2 (C-5), 154.0 (C-7), 156.4 (C-3), 185.7
(C-4 or C-1), 185.8 (C-1 or C-4). EI-MS (70 eV) m/z (%): 250 (16), 249
(100). (ESI) exact mass calcd for C12H9O6 [M ꢀ H]þ: 249.03990;
found: 249.04040 [M ꢀ H]þ.
Ref. [43], 233 ꢁC (ethyl acetate). Spectroscopic data (IR, 1H NMR, 13C
NMR, MS) in agreement with those reported in the literature [43].
5.2.5.4. 3,6,7-Trihydroxy-2-methyl-1,4-naphthoquinone (39). Accor-
ding to the protocol described in Section 5.2.1.10, 3-hydroxy-6,7-
dimethoxy-2-methyl-1,4-naphthoquinone (10.0 mg, 40 mmol) was
5.2.4.6. 3,5,6,7-Tetrahydroxy-2-methyl-1,4-naphthoquinone (35). Follo-
wing the above protocol (5.2.4.5), conducting the reaction at room
temperature, overnight, and using 10 equiv of BBr3, 34 was quantita-
tively converted to its 5,6,7-O-tridemethylated derivative, 35. Red
converted to the O-demethylated product 39. Yelloꢀw1 powder
(4.0 mg, 17.1
m
mol, 46%). Mp 190ꢀ193 ꢁC. IR (KBr, cm
) n 3756,
3630, 2360, 2341, 2160, 2043, 1646, 1576, 1325, 1187, 1154, 668. 1H
NMR (CD3COCD3) d 1.96 (s, 3H, CH3), 7.44 (s, 1H, AreH), 7.46 (s, 1H,
AreH), 9.00 (s, 1H, OH), 9.09 (s, 1H, OH), 9.31 (s, 1H, OH). EI-MS
(70 eV) m/z (%): 220 (100), 192 (50), 163 (23), 137 (49).
powder (24.0 mg, 102
m
mol, 98%). Mp 245ꢀ250 ꢁC (ethyl acetate). IR
(KBr, cmꢀ1
)
n 3488, 3302, 2921, 2850, 2582, 2463, 2078, 1744, 1609,
1583, 1555, 1506, 1448, 1404, 1388, 1349, 1314, 1275, 1212, 1168, 1103,
1049, 967, 934, 883, 795, 745, 633. 1H NMR (CDCl3):
1.94 (s, 3H, 2-
CH3), 7.12 (s, 1H, 8-H), 9.24 (s, 1H, OH), 11.4 (s, 1H, OH). 13C NMR
(CDCl3) 8.7 (CH3-2), 108.8 (C), 109.7 (CH), 121.1 (C), 126.1 (C), 137.6 (C),
d
5.2.6. The route to 3,6-dihydroxy-2-methyl-1,4-naphthoquinone
(43)
d
5.2.6.1. 2-Methyl-6-[(trifluoromethanesulfonyl)oxyl]-naphthalene
(41). To a solution of 6-hydroxy-2-methylnaphthalene (40) (0.90 g,
5.4 mmol) in CH2Cl2 (20 mL), NaH (0.65 g of a 60% powder in oil,
16.2 mmol) was added under nitrogen and the solution was stirred
for 30 min at 0 ꢁC. A solution of Tf2O (0.82 g, 5.8 mmol) in CH2Cl2
(15 mL) was added dropwise over a period of 15 min and after
stirring for 5 min, the reaction mixture was directly filtered through
a short pad of silica gel and then washed with CH2Cl2 giving a white
solid after removal of the solvent. (1.50 g, 5.3 mmol, 98%). Mp 45 ꢁC
151.6 (C), 152.5 (C), 155.0 (C), 184.3 (CO), 184.7 (CO). MS (ESI) exact
mass calcd for C11H7O6: 235.02481 [M ꢀ H]þ; found: 235.02485
[M ꢀ H]þ.
5.2.4.7. 3,6,7-Trihydroxy-5-methoxy-2-methyl-1,4-naphthoquinone
(36). In the above reaction (5.2.4.5) the 6,7-O-didemethylated
derivative of 34, compound 36, was isolated as a side product, too.
Red powder (4.0 mg, 56
(KBr, cmꢀ1
3489, 3325, 1647, 1564, 1385, 1331, 1196, 1147, 1095,
1052, 802, 746. 1H NMR (CD3COCD3):
1.93 (s, 3H, 2-CH3), 3.86 (s,
1H, 5-OCH3), 7.39 (s,1H, 8-H). 13C NMR (CD3COCD3)
8.5, 61.6,111.3,
m
mol, 15%). Mp > 340 ꢁC (ethyl acetate). IR
)
n
(petroleum ether/ethyl acetate). IR (KBr, cmꢀ1
1210, 1132, 1105, 920, 884, 822, 798, 742. 1H NMR (CDCl3):
)
n
1600, 1425, 1402,
2.53 (s,
d
d
d
3H, CH3-2), 7.32 (dd, J ¼ 8.4, 2.5 Hz, 1H, AreH), 7.41 (dd, J ¼ 8.4,
2.5 Hz,1H, AreH), 7.66 (d, J ¼ 2.5 Hz,1H, AreH), 7.70 (d, J ¼ 2.5 Hz,1H,
AreH), 7.76 (d, J ¼ 8.4 Hz,1H, AreH), 7.82 (d, J ¼ 8.4 Hz,1H, AreH).13C
116.6, 117.7, 127.8, 143.8, 149.5, 152.0, 155.3, 179.4, 184.8. EI-MS
(70 eV) m/z (%): 250 (100). (ESI) exact mass calcd for C12H9O6
[M ꢀ H]þ: 249.03990; found: 249.04040 [M ꢀ H]þ.
NMR (CDCl3)
d
21.6, 119.0, 119.1 (q, J ¼ 320.9 Hz, CF3), 119.5, 127.0,
127.8, 130.0, 130.0, 131.6, 132.8, 137.3, 146.7. EI-MS (70 eV) m/z (%):
290 (68), 157 (100), 129 (77). MS (ESI) exact mass calcd for
C12H9F3NaO3S [M þ Na]þ: 313.01167; found: 313.01168 [M þ Na]þ.
5.2.5. The route to 3,6,7-trihydroxy-2-methyl-1,4-naphthoquinone
(39)
5.2.5.1. 6,7-Dimethoxy-2-methyl-1,4-naphthoquinone (38). Follow-
ing the protocol described in Section 5.2.1.6, 6,7-dimethoxy-2-
5.2.6.2. 2-Methyl-6-[(trifluoromethanesulfonyl)oxyl]-1,4-naphthoqui
none (42). H5IO6 (2.44 g, 10.7 mmol) and CrO3 (17.8 mg, 178 mmol)
methyl-4-naphthol [32] (29.0 mg, 130 mmol) was converted to the
naphthoquinone 38. Orange-colored powder (25.0 mg, 110 mmol,
were dissolved in acetonitrile (30 mL) with vigorous stirring at 0 ꢁC.
The ester 41 (517 mg, 1.78 mmol) in acetonitrile (5 mL) was added
dropwise to the above solution. After stirring at 0 ꢁC overnight, the
reaction mixture was filtered over a short normal-phase silica gel
column, and washed quickly with CH2Cl2. After evaporation of the
solvent in vacuo, the desired product was purified by column
chromatography (petroleum ether/ethyl acetate ¼ 97: 3). Yellow
solid (199 mg, 0.62 mmol, 35%). Mp 82 ꢁC (petroleum ether/ethyl
83%). Mp 210ꢀ212 ꢁC (EtOAc); Ref. [33], 212ꢀ213 ꢁC (Ac2O). In the
literature [33], no spectroscopic data were reported. IR (KBr, cmꢀ1
) n
2924, 2850,1649,1622,1577,1512,1454,1370,1342,1308,1209,1134,
1078,1015, 985, 919, 871, 791, 742, 692, 668.1HNMR (CDCl3)
d
2.17 (d,
J ¼ 1.5 Hz, 3H, CH3), 4.02 (s, 3H, OCH3), 4.02 (s, 3H, OCH3), 6.74 (q,
J ¼ 1.6 Hz,1H, C]CH), 7.48 (s,1H, AreH), 7.52 (s,1H, AreH).13C NMR
(CDCl3) d 16.6, 56.7, 56.7, 107.8,108.2, 127.2, 127.3,135.4, 147.9, 153.5,
acetate). IR (KBr, cmꢀ1
) n 1661, 1595, 1583, 1424, 1352, 1295, 1201,
153.6, 184.8, 185.2. EI-MS (70 eV) m/z (%): 232 (100), 217 (8).
1132, 918, 866, 826, 744. 1H NMR (CDCl3):
d
2.22 (d, J ¼ 1.6 Hz, 3H,
5.2.5.2. 6,7-Dimethoxy-2-methyl-1,4-naphthoquinone-2,3-epoxide.
According to the protocol described in Section 5.2.1.8, 6,7-
CH3-2), 6.93 (d, J ¼ 1.6 Hz, 1H, H-3), 7.61 (dd, J ¼ 8.4, 2.5 Hz, 1H, H-
7), 7.94 (d, J ¼ 2.5 Hz, 1H, H-5), 8.25 (d, J ¼ 8.4 Hz, 1H, H-8). 13C NMR
dimethoxy-2-methyl-1,4-naphthoquinone
430
yellow powder (93.0 mg, 357
(EtOAc). IR (KBr, cmꢀ1
n 3057, 2946, 1678, 1573, 1514, 1459, 1444,
(38)
(100.0
mg,
(CDCl3)
d
16.7, 117.3, 118.8 (q, J ¼ 320.9 Hz, CF3), 126.5, 129.7, 131.8,
m
mol) was converted to the corresponding epoxide. Pale-
134.6, 136.0, 149.0, 153.1, 183.0, 184.0. EI-MS (70 eV) m/z (%): 320
(100), 188 (35). MS (ESI) exact mass calcd for C12H7F3O5S
[M þ Na]þ: 342.98585; found: 342.98585 [M þ Na]þ.
m
mol, 87%). Mp 145ꢀ148 ꢁC
)
1328, 1290, 1254, 1227, 1148, 1063, 1016, 984, 904, 855, 795, 767,
737, 615. 1H NMR (CDCl3)
1.72 (s,1H, CH3), 3.80 (s, 2H, CH3) 4.00 (s,
3H, OCH3), 4.01 (s, 3H, OCH3), 7.39 (s, 1H, AreH), 7.45 (s, 1H, AreH).
13C NMR (CDCl3)
15.1, 56.7, 56.8, 61.1, 61.3, 108.2, 108.9, 126.8,
d
5.2.6.3. 6-Hydroxy-2-methyl-1,4-naphthoquinone-2,3-epoxide. Acco-
rding to the protocol described in Section 5.2.1.8, the naph-
thoquinone 42 (53.0 mg, 165 mmol) was converted to the respective
d

5788
G. Bringmann et al. / European Journal of Medicinal Chemistry 46 (2011) 5778e5789
2,3-epoxide. Colorless solid (31.0 mg, 152
(MeOH/H2O). IR (KBr, cmꢀ1
3417,1694,1671,1592,1577,1321,1257,
1233, 1218, 1089, 1012, 785, 743. 1H NMR (CDCl3):
1.65 (s, 3H, CH3-
m
mol, 92%). Mp 181 ꢁC
2-methyl-1,4-naphthoquinone-2,3-epoxide (14.2 mg, 65.3
m
mol)
)
n
was converted to 45. Yellow solid (13.6 mg, 62.7 mmol, 96%). Mp
d
162ꢀ163 ꢁC (petroleum ether/ethyl acetate); Ref. [36], 174ꢀ175 ꢁC
2), 3.92 and 3.54 (s, 1H, H-3), 7.27 (dd, J ¼ 8.4, 2.5 Hz, 1H, H-7), 7.32
(benzene). In the literature [36], no spectroscopic data were re-
(d, J ¼ 2.5 Hz, 1H, H-5), 7.92 (d, J ¼ 8.4 Hz, 1H, H-8). 13C NMR (CDCl3)
ported. IR (KBr, cmꢀ1
)
n
3365, 1720, 1639, 1590, 1495, 1383, 1344,
d
15.0, 62.1, 62.4, 112.8, 122.4, 125.4, 130.8, 135.4, 163.8, 191.2, 192.3.
1316, 1271, 1231, 1070, 1024, 876, 863, 745. 1H NMR (CDCl3):
d
2.08
EI-MS (70 eV) m/z (%): 204 (100), 189 (99). MS (ESI) exact mass calcd
for C11H8NaO4 [M þ Na]þ: 227.03149; found: 227. 03148 [M þ Na]þ.
(s, 3H, CH3-2), 3.93 (s, 3H, CH3O-6), 7.19 (dd, J ¼ 8.4, 2.5 Hz,1H, H-7),
7.51 (d, J ¼ 2.5 Hz, 1H, H-5), 8.05 (d, J ¼ 8.4 Hz, 1H, H-8). 13C NMR
(CDCl3)
d 8.8 (2-CH3), 56.1 (CH3O-6), 110.1 (C-5), 120.6 (C-2), 120.9
(C-7), 126.5 (C-10), 129.2 (C-8), 131.4 (C-9), 153.1 (C-3), 163.6 (C-6),
181.6 (C-4), 184.7 (C-1). EI-MS (70 eV) m/z (%): 218 (100), 190 (26).
MS (ESI) exact mass calcd for C12H10NaO4: 241.04713 [M þ Na]þ;
found: 241.04718 [M þ Na]þ.
5.2.6.4. 3,6-Dihydroxy-2-methyl-1,4-naphthoquinone (43). Accord-
ing to the protocol described in Section 5.2.1.9, the epoxide
prepared in 5.2.6.3. (4.3 mg, 12.7 mmol) was converted to the cor-
responding naphthoquinone, 43. Yellow solid (4.2 mg, 12.5 mꢀm1ol,
98%). Mp > 340 ꢁC (petroleum ether/ethyl acetate). IR (KBr, cm
) n
5.2.7. Extraction and isolation
3280, 1666, 1651, 1631, 1573, 1375, 1303, 1258, 1062, 1014, 887, 795.
1H NMR (CDCl3):
d
1.99 (s, 3H, CH3-2), 7.07 (dd, J ¼ 8.4, 2.5 Hz,1H, H-
Lyophilized cell cultures [12] of T. peltatum (35.2 g) were
ground and extracted with CH2Cl2/CH3OH (1:1). The extract was
condensed under reduced pressure to give 6.6 g of a crude residue,
which was subjected to ion-exchange column to remove naphthyl
isoquinoline alkaloids, followed by normal-phase chromatography
affording three main naphthoquinones: droserone (2) (50.0 mg),
dioncoquinone A (5) (110 mg), and dioncoquinone B (6) (44.0 mg),
and further by preparative HPLC permitting the isolation of the
four further naphthoquinones, 1.0 mg of dioncoquinone C (7)
(retention time 10.9 min), 0.8 mg of dioncoquinone D (8) (reten-
tion time 18.9 min), 1.6 mg of dioncoquinone E (9) (retention time
11.2 min), and 3.0 mg of 8-hydroxydroserone (10) (retention time
11.0 min).
7), 7.37 (d, J ¼ 2.5 Hz, 1H, H-5), 7.89 (d, J ¼ 8.4 Hz, 1H, H-8). 13C NMR
(CDCl3)
d 8.5 (2eCH3), 112.9 (C-5), 121.0 (C-2), 121.6 (C-7), 126.1 (C-
10), 129.7 (C-8), 133.6 (C-9), 156.4 (C-3), 163.5 (C-6), 182.3 (C-4),
186.6 (C-1). EI-MS (70 eV) m/z (%): 204 (100), 176 (27). MS (ESI)
exact mass calcd for C11H8NaO4 [M þ Na]þ: 227.03149; found: 227.
03148 [M þ Na]þ.
5.2.6.5. 6-Hydroxy-2-methyl-1,4-naphthoquinone (44). To a solution
of the trifluoromethanesulfonic ester 42 (150 mg, 470
dioxane (2 mL), 10% aqueous Et4NOH solution (689 mg, 940
673 L) was carefully added at ambient temperature. The mixture was
mmol) in
mmol,
m
stirred for 30 min, diluted with CH2Cl2 (10 mL) and water (10 mL), and
neutralized with 1 N aqueous HCl solution. The organic layer was
dried over anhydrous Na2SO4, concentrated in vacuo and purified by
5.2.7.1. 3,5,6-Trihydroxy-7-methoxy-2-methyl-1,4-naphthoquinone
(dioncoquinone C, 7). Yellow solid. Mp > 340 ꢁC (ethyl acetate). All
spectroscopic data (IR, 1H NMR, 13C NMR, MS) identical with the
synthetic material in 5.2.4.5.
column chromatography. Yellow solid (44.0 mg, 235
175 ꢁC (petroleum ether/ethyl acetate). IR (KBr, cmꢀ1
1650, 1594, 1573, 1457, 1350, 1329, 1267, 1235, 1203, 1134, 887, 848,
mmol, 50%). Mp
)
n
3361, 1662,
1
692. H NMR (CD3OCD3):
d
2.13 (d, J ¼ 1.6 Hz, 3H, CH3-2), 6.82 (d,
5.2.7.2. 5,6-Dihydroxy-3,7-dimethoxy-2-methyl-1,4-naphthoquinone
J ¼ 1.6 Hz, 1H, H-3), 7.22 (dd, J ¼ 8.4, 2.5 Hz, 1H, H-7), 7.39 (d,
(dioncoquinone D, 8). Yellow solid. Mp > 340 ꢁC 1H NMR (CDCl3):
J ¼ 2.5 Hz, 1H, H-5), 7.95 (d, J ¼ 8.4 Hz, 1H, H-8). 13C NMR (CD3OCD3)
d
2.05 (s, 3H, 2-CH3), 4.01 (s, 3H, 7-OCH3), 4.05 (s, 3H, 3-OCH3), 7.26
d
16.4 (2-CH3), 112.4 (C-5), 121.3 (C-7), 125.9 (C-10), 130.0 (C-8), 135.6
(s, 1H, 8-H), 11.82 (s, 1H, 5-OH). 13C NMR (CDCl3)
d
9.6 (CH3-2), 58.0
(C-9), 135.9 (C-3), 149.4 (C-2), 163.4 (C-6), 184.5 (C-1), 185.5 (C-4). EI-
MS (70 eV) m/z (%): 188 (100), 160 (23). MS (ESI) exact mass calcd for
C11H7O3 [M ꢀ H]þ: 187.04006; found: 187.04707 [M ꢀ H]þ.
(CH3O-7), 61.3 (CH3O-3), 103.6 (C-8), 110.1 (C-10), 124.4 (C-9), 133.3
(C-2), 138.2 (C-6), 149.2 (C-5), 151.3 (C-7), 157.1 (C-3), 184.4 (C-1),
185.6 (C-4). (ESI) exact mass calcd for C13H11O6 [M ꢀ H]þ:
263.05557; found: 263.05610 [M ꢀ H]þ.
5.2.6.6. 6-Methoxy-2-methyl-1,4-naphthoquinone-2,3-epoxide. K2CO3
(98.0 mg, 710
methyl-1,4-naphthoquinone-2,3-epoxide (29.0 mg, 142
acetone (3 mL) at 0 ꢁC. The mixture was magnetically stirred for
30 min, then dimethylsulfate (89.5 mg, 67.4 L, 710 mol)wasinjected
m
mol) was added to the solution of 6-hydroxy-2-
5.2.7.3. 3,5,7-Trihydroxy-6-methoxy-2-methyl-1,4-naphthoquinone
(dioncoquinone E, 9). Yellow solid. Mp > 340 ꢁC 1H NMR (CD3OD):
mmol) in
d
1.96 (s, 3H, 2-CH3), 3.91 (s, 3H, 6-OCH3), 7.09 (s, 1H, 8-H). 13C NMR
m
m
(CD3OD) d 8.5 (CH3-2), 61.1 (CH3O-6), 109.1 (C-10), 110.2 (C-8), 121.2
into the flask via a syringe and stirred for 3 h. Thereaction mixturewas
quenched by addition of water (20 mL), neutralized with 1 N aqueous
HCl solution, and the aqueous layer was extracted with CH2Cl2
(3 ꢂ 20 mL). The combined organic layers were dried with Na2SO4,
concentrated to dryness, purified by column chromatography to
(C-2), 130.2 (C-9), 139.7 (C-6), 157.0 (C-3), 157.3 (C-5), 158.6 (C-7),
184.9 (C-4), 185.8 (C-1). (ESI) exact mass calcd for C12H9O6
[M ꢀ H]þ: 249.03990; found: 249.04040 [M ꢀ H]þ.
5.2.7.4. 8-hydroxydroserone (10). Red solid. Mp 193 ꢁC (CHCl3);
Ref. [14], 193 ꢁC (CHCl3). All spectroscopic data (IR, 1H NMR, 13C
NMR, MS) were identical with those in the literature [14].
afford the desired epoxide as a colorlessꢀs1olid (30.3 mg, 139.2
mmol,
98%). Mp 89 ꢁC (MeOH/H2O). IR (KBr, cm
) n 1684, 1592, 1494, 1433,
1294,1232,1192,1057,1026, 948, 856, 780, 740.1H NMR(CDCl3):
d 1.71
(s, 3H, CH3-2), 3.83 (s, 1H, H-3), 3.91 (s, 3H, CH3O-6), 7.21 (dd, J ¼ 8.4,
5.3. Biological tests
2.5Hz,1H, H-7), 7.37 (d, J¼ 2.5 Hz,1H, H-5), 7.97(d, J¼ 8.4 Hz,1H, H-8).
13C NMR (CDCl3)
d 15.0 (2-CH3), 56.1 (CH3O-6), 61.4 (C-2), 61.6 (C-3),
The effects of dioncoquinones B and C and related naph-
thoquinones on cell survival were analyzed either in non-
malignant cells (mononuclear cells derived from the peripheral
blood of healthy donors) or in malignant cells of the human
multiple myeloma cell line INA-6 [44]. Apoptotic and viable cell
fractions were determined by staining with annexin V-FITC and
propidium iodide (PI) according to the manufactures’ instructions
(Bender MedSystems, Vienna, Austria). In brief, cells were washed
110.0 (C-5), 121.7 (C-7),125.5 (C-10), 130.1 (C-8), 134.3 (C-9), 164.7 (C-
6), 190.8 (C-1), 192.0 (C-4). EI-MS (70 eV) m/z (%): 218 (100), 203 (82),
119 (51). MS (ESI) exact mass calcd for C12H10NaO4: 241.04713
[M þ Na]þ; found: 241.04702 [M þ Na]þ.
5.2.6.7. 3-Hydroxy-6-methoxy-2-methyl-1,4-naphthoquinone (45).
According to the protocol described in Section 5.2.1.9, 6-methoxy-

G. Bringmann et al. / European Journal of Medicinal Chemistry 46 (2011) 5778e5789
5789
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2.5 mL annexin V-FITC and 1 mg/mL PI, subsequently diluted with
300 mL binding buffer and analyzed by flow cytometry (FACSCali-
bur/CELLQuest; Becton Dickinson, Heidelberg, Germany). In early
stages of the apoptotic process phosphatidylserine translocates
from the internal to the external membrane, and can be detected by
annexin V-FITC. Cells in a late apoptotic stage lose their membrane
integrity and the DNA-binding agent PI can be incorporated. Thus,
viable cells are negative for both, annexin V-FITC and PI.
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