Synthesis and biological evaluation of new derivatives of emodin

Article abstract of DOI:10.1016/j.bmc.2004.08.024

Synthesis and characterization of new emodin derivatives. Cytostatic potential and binding to multi-drug-resistance transporters were evaluated. Drugs containing an anthraquinone moiety such as daunorubicin (Daunoblastin) and mitoxantrone (Onkotrone) constitute some of the most powerful cytostatics. They suppress tumor growth mainly by intercalation into DNA and inhibition of topoisomerase II, and are suspected to generate free radicals leading to DNA strand scission. We established a novel strategy for obtaining new highly functionalized derivatives of emodin (1,3,8-trihydroxy-6- methyl-anthraquinone). Using emodin, DIB, and an appropriate amine as starting materials, we obtained a wide range of emodin-related structures by one-pot synthesis. Several of these derivatives showed stronger cytotoxic and cytostatic activity than emodin. In particular, compound 6 was highly effective on the HepG2 tumor cell line, but did not show any cytotoxicity on normal hepatocytes. In addition to this favorable feature, compound 6 revealed interesting binding properties to a recombinant fragment of the multi-drug-resistance transporter, pgp, and reversed the multi-drug-resistance phenotype of H4-II-E cells, thus making this compound a promising potential anti-tumor drug.

Full text of DOI:10.1016/j.bmc.2004.08.024

Bioorganic & Medicinal Chemistry 12 (2004) 5961–5971
Synthesis and biological evaluation of new derivatives of emodin
b
a
Lars Teich, Katja Scarlett Daub, Vera Kr u¨ gel, Ludwig Nissler,
a
b
b
Rolf Gebhardt and Kurt Eger
a,
*
a
Institut f u¨ r Pharmazie, Fakult a¨ t f u¨ r Biowissenschaften, Pharmazie und Psychologie, Universit a¨ t Leipzig, Br u¨ derstraße 34,
4103Leipzig, Germany
0
Institut f u¨ r Biochemie, Medizinische Fakult a¨ t, Universit a¨ t Leipzig, Liebigstraße 16, 04103Leipzig, Germany
b
Received 14 October 2003; accepted 13 August 2004
Available online 15 September 2004
Dedicated to Professor Reinhard Trosch u¨ tz, Universit a¨ t Erlangen, on the occasion of his 60th birthday
ꢀ
ꢀ
Abstract—Drugs containing an anthraquinone moiety such as daunorubicin (Daunoblastin ) and mitoxantrone (Onkotrone ) con-
stitute some of the most powerful cytostatics. They suppress tumor growth mainly by intercalation into DNA and inhibition of
topoisomerase II, and are suspected to generate free radicals leading to DNA strand scission. We established a novel strategy
for obtaining new highly functionalized derivatives of emodin (1,3,8-trihydroxy-6-methyl-anthraquinone). Using emodin, DIB,
and an appropriate amine as starting materials, we obtained a wide range of emodin-related structures by one-pot synthesis. Several
of these derivatives showed stronger cytotoxic and cytostatic activity than emodin. In particular, compound 6 was highly effective on
the HepG2 tumor cell line, but did not show any cytotoxicity on normal hepatocytes. In addition to this favorable feature, com-
pound 6 revealed interesting binding properties to a recombinant fragment of the multi-drug-resistance transporter, pgp, and
reversed the multi-drug-resistance phenotype of H4-II-E cells, thus making this compound a promising potential anti-tumor drug.
ꢁ
2004 Elsevier Ltd. All rights reserved.
1. Introduction
OH
O
OH
Emodin (I) occurs in different plants (rhubarb, aloe) and
is a widespread dye in fungi and lichens. It belongs to
the anthraquinone group along with daunorubicin (II)
and mitoxantrone (III), which constitute some of the
most powerful cytostatics (Fig. 1).
HO
CH3
O
l
H
N
O
O
OH
OH
O
OH
O
O
HN
HN
OH
OH
1
Emodin itself is a tumor cell-growth inhibitor. Emodin
2
CH3
3
OH
possesses diuretic and vasodilatatoric effects as well as
6,7
antibiotic, anti-viral, and anti-neoplastic activity.
4
,5
8,9
1
0
H CO
O
O
It is also supposed to sensitize cancer cells. Thus, emo-
din sensitizes HER/neu-overexpressing cancer cells to
chemotherapeutic agents such as cisplatin, doxorubicin,
3
OH
N
H
CH3
HO
1
1
etoposide, and paclitaxel. Furthermore, emodin in-
duces apoptosis in CH27 and H460 lung carcinoma cell
lines due to an increase in cytochrome c of the cytosolic
fraction and activation of caspase-3, caspase-9, and
caspase-8. PKC isoenzyme expression is involved in
emodin-induced apoptosis of CH27 and H460 lung
carcinoma cells, and appears to be associated with the
NH2
ll
lll
Figure 1. Emodin (I), daunorubicin (II), and mitoxantrone (III).
increased expression of cellular Bak and Bax proteins,
whereas no modulation of endogenous Bcl-XL protein
expression has been observed.
1
2,13
We established a
*
Corresponding author. Tel.: +49 341 9736800; fax: +49 341
736709; e-mail: eger@rz.uni-leipzig.de
novel strategy to obtain new derivatives of emodin (1,3,8-
trihydroxy-6-methyl-anthraquinone). These derivatives
9
0
968-0896/$ - see front matter ꢁ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.08.024

5962
L. Teich et al. / Bioorg. Med. Chem. 12 (2004) 5961–5971
1
5
were screened against the HepG2 hepatoblastoma cell
line and normal hepatocytes for their cytotoxic and
cytostatic properties and for their binding capacity to
a recombinant fragment of the multi-drug-resistance
transporter, pgp.
the OH-3 group (physcione) do not react. Some
amines underwent further reactions: Ring-like amines
as piperidine not only gave rise to the primary formed
4-(N-piperidinyl) derivative 7, but also to the dihydro-
oxazole derivative 10 at prolonged reaction times. The
smaller analogue pyrrolidine only resulted in the
dihydrooxazole derivative 9. We assume that in this
cases a second DIB molecule is used for the oxidation
of the amine moiety. When 1,2-diamines were used, ann-
elated tetrahydropyrazine derivatives were formed. So
ethylenediamine gave 4-[N-(2-aminoethyl)-amino] deriv-
ative 3, which was converted to tetrahydropyrazine 11 at
2. Chemistry
Compounds 1–13 were synthesized using a novel syn-
thetic strategy (Figs. 2–4). Emodin (I) was reacted with
DIB in the presence of an appropriate amine. Under this
conditions emodin underwent an oxidizing amination by
substitution of the hydrogen in position 4 by an amine-
N, yielding 4-amino-emodin derivatives in one step.
Depending on the structure of the used amine further
reactions occurred, broadening the range of structures.
OH
O
5
OH
4
OH
O
OH
4
6
6
7
3
2
7
5
3
2
12
12
CH3
CH3
8
O
9
1
8
O
1
The poor solubility of emodin and DIB in common sol-
vents was circumvented by using the amine itself as the
solvent. Depending on the amine, the reaction times ran-
ged from a matter of hours to days at room temperature.
Reactions could be easily followed by TLC, because of
the big differences of color and luminescence properties
of the reaction products (Table 1). Temperature increase
did not shorten reaction times, but did lead to lower
yields in most cases. The structures thus obtained can
be divided in three classes: amines (Fig. 2), annelated
dihydrooxazole derivatives (Fig. 3) and annelated tetra-
hydropyrazine derivatives (Fig. 4). 4-Aminoemodines 3–
N
O
N
O
12
11
9
1
0
10
11
9
10
Figure 3. Synthesized 4-amino-emodin derivatives (annelated dihydro-
oxazole derivatives).
OH
O
OH
8
OH
O
OH
9
6
7
5
7
9
6
CH3 HN5
4
8
10
11
CH3
10
13
12
HN4
11
12
8
were formed by converting emodin (I) with DIB in the
1NH
14NH
1
O
O
3
presence of the appropriate amine, similar to reactions
with acetophenone derivatives described by Wells
2
11
3
2
12
1
4
et al. We assume that in our case the primary step is
the addition of DIB at OH-3 followed by amine attach-
ment with dearomatization. After splitting of iodobenz-
ene and two molecules of acetic acid rearomatization
occurs (Scheme 1).
OH
NH
O
OH
H C
3
N
CH3
O
1
3
This is strengthened by the observation that similar
compounds lacking the OH-3 group (chrysophanol,
chrysazine) or displaying a methoxy group instead of
Figure 4. Synthesized 4-amino-emodin derivatives (annelated tetra-
hydropyrazine derivatives).
OH
O
9
OH
8
OH
O
OH
OH
NH
O
OH
OH
NH
O
O
OH
1
2
3
7
6
10
HO
CH3
HO
CH3
HO
CH3
HO
CH3
4
5
NH2
O
NH
O
O
1'
2'
1
2
3
4
HO
H N
N
2
H C
CH3
3
OH
NH
O
O
OH
OH
O
OH
OH
O
OH
OH
O
OH
HO
CH3
HO
CH3
HO
CH3
HO
CH3
NH
O
N
4'
6'O
N
O
O
2
'
1
'
2
'
1' 6'
2'
3'
5'
3'
5'
4'
3
'
5
6
7
8
NH2
Figure 2. Synthesized 4-amino-emodin derivatives (amines).

L. Teich et al. / Bioorg. Med. Chem. 12 (2004) 5961–5971
5963
Table 1. UV–vis data of synthesized emodin derivatives (in methanol)
Compound
kmax in nm (e)
3
3
4
220 (20 * 10 ), 274 (17 * 10 ), 546
(
3
3
12 * 10 ), 587 (14 * 10 )
3
3
6
225 (16 10 ), 261 (18 10 ), 554
* *
3
3
(
14 * 10 ), 595 (16 * 10 )
3
3
3
9
223 (20 * 10 ), 258 (23 * 10 ), 520 (9 * 10 )
3
3
3
11
226 (18 * 10 ), 277 (17 * 10 ), 514 (7 * 10 ),
3
50 (16 10 ), 592 (18 10 )
3
5
*
*
Figure 5. X-ray crystallographic structure of 13.
H
N
OH
O
O
OH
OH
H
O
O
OH
R'
R''
1
plex mixtures of very hydrophilic compounds, no main
components could be observed. Due to the 1,4- and
PhI(OAc)2
3
HO
CH3
- AcOH
O
CH3
1,2-hydro-quinone substructure all compounds are quite
Ph
I
susceptible to degradation and should be handled under
mild conditions such as low temperatures, fast isolating
and cleaning procedures, and short storage times for
solutions prepared. However, using this synthetic strat-
egy, it is possible now to obtain a wide range of highly
substituted emodin derivatives in only one step!
AcO
Emodin
H N
R' R''
-
-
AcOH
PhI
OH
O
O
OH
OH
O
OH
To alter the solubility and the pharmacokinetic proper-
ties amides 14–16 were synthesized by reaction of com-
pound 5 with the appropriate carbonic acid anhydride
in dry methanol, from which the crude product can be
isolated by filtration (Fig. 6).
HO
CH3
O
CH3
N
H
N
O
R'
R''
R' R"
Scheme 1. Possible mechanistic formation pathway of 4-amino-emo-
din derivatives.
All 4-amino-emodin derivatives 1–16 show strong color
intensity (see Table 1 for example). What has to be con-
sidered using fluorescence-based biological assays.
prolonged reaction times. The space-demanding trans-
1
,2-diaminocyclohexane gave rise to 12. While due to
sterical reasons 1,3-diamines as 5 did not cyclize. An
unexpected reaction was the cleavage of C–O– and C–
NH– bonds by DIB: Similar to piperidine morpholine
yield the 4-(N-morpholinyl) derivative 8, but prolonged
reaction times led to 4-[N-(2-hydroxyethyl)-amino]-emo-
din 2 by C–O– bond cleavage. When piperazine was
used, the C–NH– bond was cleaved leading to the pri-
mary amine 3, which was converted to tetrahydropyr-
azine derivative 11 as described above for the reaction
of ethylenediamine. The same reaction occurred when
N-methyl-piperazine was used and the N-methyl-tetra-
hydropyrazine derivative 13 was obtained, no primary
product could be isolated in considerable amounts.
Extensive structure investigations using NMR-coupling
techniques were performed to confirm this product.
Due to the lack of hydrogens and the many quaternary
carbon atoms we still had doubts concerning the substi-
tution pattern. Finally, X-ray diffraction analysis gave
Synthesis of tetranitro-emodin derivatives 17 and 18
were carried out with good yields using standard proce-
dures (H SO /HNO ). Acetylation of the steric hindered
2
4
3
OH
O
O
OH
OH
NH
O
O
OH
HO
CH3
HO
CH3
NH
2'
1'
3'
NH
14
NH
1"
15
O
O
6"
CH3
5"
2"
OH
O
O
OH
4"
3"
1
6
definitive confirmation of the substructure (Fig. 5).
HO
CH3
In all cases long reaction times or elevated temperatures
led to degradation and formation of 4-amino-emodine 1
in traces. Emodin and an excess of DIB in boiling piper-
idine for 10h is a possible preparation of 1. Due to the
incomplete consumption of emodin and the formation
of highly oxidized degradation products middle and
NH
NH
16
O
6"
5"
4
"
1
7
low yields were observed (13–76%). Therefore, remain-
ing emodin was recovered by chromatographic workup.
The degradation products were removed by extractive
workup. Analysis of the washing waters showed com-
HO
1"
3"
2"
O
Figure 6. Synthesized amides of compound 5.

5964
L. Teich et al. / Bioorg. Med. Chem. 12 (2004) 5961–5971
OH
O
OH
8
OH
O
OH
120
00
1
O N
NO2
O N
NO2
2
9
2
2
3
7
6
1
10
HO
CH3
4
5
Hepatocytes
NO2
O
NO2
NO2
O
NO2
80
1
7
18
60
O
O
H C
O
O
O
O
CH3
3
40
O N
NO2
2
20
HO
CH3
HepG2
15
NO2
NO2
0
0
5
10
20
1
9
Concentration (µM)
Figure 7. Synthesized tetranitro-emodin derivatives.
Figure 8. Cytotoxicity of 6 compared for HepG2 cells (closed circles)
and rat hepatocytes (open circles) in the relevant concentration range.
Cytotoxicity was determined by MTT assay. Values are presented as
the mean ± SD of three independent determinations.
1
1
8 in acetic anhydride produced diacetylated compound
9 (Fig. 7).
Cytotoxic influence of these compounds on primary rat
hepatocytes and HepG2 cells was considerably diverse
3. Biological results
(
Table 2). Three compounds, 1, 6, and 10, were respec-
tively 2.5, 5.1, and 2.4 times as cytotoxic to HepG2
hepatoblastoma cells as emodin based on EC50 values.
Most remarkably, normal primary hepatocytes were al-
most unaffected (by a factor of at least 0.04-fold) by
compound 6 (Fig. 8), whereas the other two also acted
somewhat more vigorously on these cells than did
emodin.
Cytostatic potential of the 4-amino-emodin and tetrani-
tro-emodin derivatives was determined by measuring
their cytotoxicity against normal and transformed
hepatocytes and their inhibition of thymidine incorpora-
tion in growing cells. Cytotoxicity was determined using
the MTT assay. Since some of the compounds showed
considerable color and/or fluorescence, interference
with this assay was unavoidable; however, appropriate
measures were taken to obtain correct results. Proper
determination of EC50 values was only impossible
under conditions of limited solubility in culture medium
Several compounds, 2, 7, 8, 16, 18, and 19, showed the
reverse characteristic and were slightly more cytotoxic
on the normal than on the transformed hepatocellular
phenotype (Table 2). In these cases, however, only com-
pound 8 was generally more cytotoxic than emodin.
Modifications in the emodin structure leading to com-
pounds 3, 4, 5, 9, 11, 12, and 13 completely destroyed
the cytotoxic potential of emodin. There is no apparent
simple structure–function relationship. In particular, no
clear distinction could be made between 4-amino-emo-
din and tetranitro-emodin derivatives. Among 4-amino-
emodin derivatives 1–16, voluminous or possibly
charged structural additions to the 4-amino group
seemed unfavorable. Whether the 4-amino group used
either as a primary or secondary amine might retain
the possibility of forming a hydrogen bond in order to
enhance efficacy remains to be determined. A similar
ranking as for the cytotoxic effect was apparent with re-
(
Table 2).
Table 2. Cytotoxicity of synthesized emodin derivatives on primary rat
hepatocytes and HepG2 cells
Compound
Limit
of solubility (lM)
Cytotoxicity EC50 values
(lM)
Hepatocytes
HepG2 cells
Emodin
>400
>400
38.2 ± 3.7
11.7 ± 0.4
32.4 ± 1.1
>400
42.9 ± 1.1
17.3 ± 0.9
ꢀ70
1
2
3
4
5
6
7
8
9
ꢀ400
200–400
ꢀ500
200–400
ꢀ200
>800
>400
140 ± 1.5
>400
>200
>200
ꢀ300
3
8.4 ± 0.5
88.6 ± 1.7
34.1 2.0
>200
spect to the inhibition of [ H]-thymidine incorporation
into HepG2 cell DNA (Table 3).
74.7 ± 1.6
13.8 ± 1.3
360 ± 20
35–40
>400
>400
Of course, inhibition was generally observed at lower
concentrations than the cytotoxic effect. Some com-
pounds, namely 1, 2, 6, and 10, exerted a stronger inhi-
bition than emodin I. Again, compound 6 appeared to
be the most effective growth inhibitor at approximately
five times more effective. When compared to mitoxan-
throne and losoxanthrone, the cytotoxic and cytostatic
10
11
12
13
14
15
16
17
18
19
>800
>400
17.9 ± 0.7
>400
>400
>400
>400
ꢀ400
ꢀ400
>400
>400
>400
>400
>400
>400
>400
>400
ꢀ200
ꢀ300
200–400
ꢀ200
>200
12.7 ± 0.3
>200
75 ± 8
1
8,19
>200
ꢀ150
effects of compound 6 were stronger, too.
Since this
compound does not affect the normal hepatocellular
phenotype, a feature that is often overlooked in searches
53 ± 8
ꢀ150

L. Teich et al. / Bioorg. Med. Chem. 12 (2004) 5961–5971
5965
3
Table 3. Effects of synthesized emodin derivatives on [ H]-thymidine
Emodin binding to the recombinant cytosolic fragment
of pgp revealed an EC50 value of 18.9 and a maximal
quenching of 79%. Most derivatives were less effective
than emodin in terms of EC50 values, although some ex-
erted a higher maximal quenching of 100%. Apparently,
tetranitro derivatives 17, 18, and 19 were more effective
than 4-amino derivatives 1–16. In particular, derivative
incorporation into HepG2 cell DNA
3
Compound
H-Thymidine incorporation into
DNA (% control)
1
0lM
40lM
Emodin
86.9 ± 3.6
15.6 ± 1.2
81.5 ± 0.1
91.5 ± 2.0
90.8 ± 3.1
86.3 ± 1.7
35.5 ± 3.4
65.6 ± 0.7
56.9 ± 0.7
91.4 ± 0.9
77.2 ± 1.1
83.4 ± 0.9
99.4 ± 1.2
103 ± 4
22.5 ± 3.0
10.2 ± 0.9
11.3 ± 0.1
77.3 ± 1.8
28.2 ± 1.2
42.8 ± 1.8
4.9 ± 0.1
1
2
3
4
5
6
7
8
9
19 was almost twice as strong as emodin, and addition-
ally led to 100% quenching. Therefore, this compound
may represent a potentially interesting pgp inhibitor,
but this would have to be substantiated in further exper-
iments. Interestingly, derivative 6 also turned out to
bind slightly better than emodin. Thus, in addition to
its favorable cytostatic features, this derivative also
seems to retain the potential to inhibit pgp transport-
ers. This was demonstrated using H4-II-E cells, which
show a multi-drug-resistance phenotype preventing
cellular accumulation of doxorubicin. In the presence
of 40lM compound 6 or emodin, this phenotype was
reversed and accumulation of doxorubicin amounted
to 31.5 ± 1.7% and 64.0 ± 3.9% (N = 3), respectively.
Cyclosporine A (1lM) served as control and its effect
was set 100%. The combination of these properties is
unusual and renders this compound a very promising
potential anti-tumor drug.
22.7 ± 0.2
21.2 ± 0.1
30.4 ± 0.8
10.9 ± 0.3
84.5 ± 1.9
98.9 ± 0.1
101 ± 1
10
11
12
13
14
15
16
17
18
19
86.5 ± 3.7
62.9 ± 2.0
95.1 ± 1.6
101 ± 2
72.8 ± 2.7
20.1 ± 1.0
65.1 ± 0.7
73.8 ± 3.3
35.5 ± 0.2
19.9 ± 0.2
79.2 ± 0.8
47.3 ± 0.7
for new cytostatic drugs (!), this compound is an inter-
esting candidate for such a drug, and worth for a further
characterization through in vitro and in vivo studies.
Further evaluation of these emodin derivatives was
made with respect to possible binding and inhibition
of multi-drug-resistance transporters. In order to facili-
tate these studies, interaction between these compounds
and a recombinant cytosolic C-terminal fragment of pgp
representing the second ATP-binding loop was meas-
ured by quenching the intrinsic fluorescence (Table 4).
4. Conclusion
A novel synthetic approach to highly functionalized
derivatives of emodin facilitates preparation of highly
functionalized emodin derivatives in only one step.
Using emodin, DIB, and an appropriate amine as start-
ing materials, we were able to produce a wide range of
emodin-related structures in a high regioselective man-
ner by one-pot synthesis. Some of these emodin deriva-
tives showed stronger cytotoxic and cytostatic activity
than emodin and/or bound more effectively to the sec-
ond ATP-binding loop of pgp. Most interestingly, com-
pound 6 discriminated well between hepatoma cells and
primary hepatocytes and retained the capacity to reverse
the multi-drug-resistance phenotype. This compound,
therefore, features unmatched properties of a potential
anti-tumor drug.
Table 4. Interaction of synthesized emodin derivatives with recomb-
inant pgp
Compound
Fluorescence quenching of recombinant
pgp
EC50 (lM)
Max. quenching (%)
Emodin
18.9 ± 5.2
19.1 ± 2.4
27.5 ± 4.7
33.8 ± 17.9
24.8 ± 5.4
24.8 ± 4.5
14.0 ± 3.0
30.8 ± 9.1
9.1 ± 2.7
79.0
100.0
92.0
1
2
3
4
5
6
7
8
9
5. Experimental
100.0
77.3
5.1. Chemistry
100.0
77.9
96.0
Melting points were measured in open capillaries in an
1
13
oil bath and are uncorrected. H and C nuclear mag-
netic resonance spectra were obtained with a Varian
Gemini-300 spectrometer. The chemical shifts were
recorded in parts per million (ppm) with the chemical
shifts of the remaining protons of the deuterated sol-
vents serving as internal standards. H, H-COSY,
HMBC, HMQC, and NOE experiments were acquired
using a Bruker DRX instrument (600MHz). High-reso-
lution mass spectra were measured on a 7T APEX II
FT-ICR-ESI mass spectrometer (Bruker Daltonics).
Colorimetric measurements were carried out with a
Shimadzu-UV-Vis spectrometer. IR spectra were
56.0
100.0
75.0
41.3 ± 13.4
26.5 ± 5.4
37.2 ± 14.2
18.7 ± 6.8
26.8 ± 5.2
17.7 ± 1.3
19.1 ± 3.0
17.1 ± 2.5
16.0 ± 1.0
12.0 ± 0.7
9.5 ± 0.4
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
100.0
47.0
90.2
100.0
100.0
100.0
100.0
100.0
98.8

5966
L. Teich et al. / Bioorg. Med. Chem. 12 (2004) 5961–5971
obtained with a Perkin–Elmer FT-IR PC16 spectrome-
ter. Thin-layer chromatography was performed on Silica
Gel 60 F254 plates (Merck) and visualized by color and
under UV light (254 and 366nm). Merck Silica Gel 60
product was purified by adsorptive filtration on silica gel
(short column), the first eluate (ethyl acetate) contained
traces of compound 11, the second eluate (methanol)
compound 3. Violet crystals (80mg, 24%), mp 178–
(0.040–0.063mm) was used for column chromato-
graphy.
185ꢂC; R = 0.2 (ethyl acetate/ethanol/water 20:2:1) vio-
f
ꢁ
1
let, red fluorescence; IR (KBr) cm : 3421, 1600, 1544,
1
499, 1404, 1358, 1319, 1283, 1209, 753; H NMR
1
5
5
.2. Synthesis of 4-amino-emodin derivatives
(methanol-d ) d 2.42 (s, 3H, CH -6), 3.19 (t, 2H,
4
3
3
0
3
J = 5.7Hz, CH -2 ), 4.14 (t, 2H, J = 5.7Hz, CH -1 ),
0
2
2
.2.1. 4-Amino-emodin (1). (Diacetoxyiodo)benzene
5.96 (s, 1H, H-2), 6.86 (s, 1H, H-7), 7.56 (s, 1H, H-5);
H NMR (DMF-d ) d 2.39 (s, 3H, CH -6), 2.58 (m,
1
(
(
370mg, 1.15mmol) was added to a solution of emodin
270mg, 1.0mmol) in piperidine (30mL) and refluxed
7
3
0
0
2H, CH -2 ), 4.26 (m, 2H, CH -1 ), 5.69 (s, 1H, H-2),
2
2
for 10h. Piperidine was removed in vacuo. The residue
was extracted with ethyl acetate (3 · 100mL). The or-
ganic layer was washed in 0.05M HCl (150mL) and
water (3 · 100mL) and evaporated. The crude product
was purified by column chromatography in two steps
first on silica gel (petroleum ether/acetone/ethyl acetate
6.85 (s, 1H, H-7), 7.60 (s, 1H, H-5), 12.21 (br s, 1H,
NH-1), 13.62 (br s, 1H, OH), 14.88 (br s, 1H, OH);
1
3
0
C NMR (DMF-d ) d 21.8 (CH -6), 42.5 (CH -2 ),
7
3
2
0
44.6 (CH -1 ), 101.3 (C-9a), 106.1 (C-2), 116.5 (C-8a),
117.9 (C-5), 119.1 (C-4a), 119.9 (C-7), 121.7 (CNH-4),
135.9 (C-10a), 143.4 (CCH -6), 149.8 (C-3), 161.7
2
3
1
:1:1), then on Sephadex LH-20 with methanol to give
compound 1. Violet powder (57mg, 20%), mp >
50ꢂC; R = 0.6 (ethyl acetate/ethanol/water 20:2:1)
(COH-8), 169.0 (COH-1), 176.9 (CO-9), 180.6 (CO-
10); HRMS calcd for C H N O : 329.11320, found
329.11335.
+
1
7
17
2
5
3
f
ꢁ
1
pink, orange fluorescence; IR (KBr) cm : 3422, 2993,
1
1
718, 1685, 1588, 1508, 1387, 1295, 1264, 1206;
H
5.2.4. 4-[N-(2-Dimethylaminoethyl)-amino]-emodin (4).
(Diacetoxyiodo)benzene (354mg, 1.1mmol) was added
to a solution of emodin (270mg, 1.0mmol) in N,N-
dimethylethylenediamine (40mL) and stirred for 4days
at room temperature. The reaction was monitored by
TLC. The solution was poured into a mixture of ice
(200g), 10M HCl (60mL), neutralized with saturated
NMR (methanol-d ) d (ppm) 2.47 (s, 3H, CH ), 6.50
4
3
1
s, 1H, H-2), 7.01 (s, 1H, H-7), 7.64 (s, 1H, H-5); H
(
NMR (DMSO-d ) 2.41 (s, 3H, CH ), 6.25 (s, 1H, H-
6
3
2
1
1
1
), 6.96 (s, 1H, H-7), 7.52 (s, 1H, H-5), 12.40 (OH),
3.48 (OH); C NMR (acetone-d ) d 21.5 (CH -6),
1
3
6
3
01.0 (C-9a), 106.2 (C-4a), 106.8 (CH-2), 114.6 (C-8a),
19.3 (CH-5), 121.8 (CH-7), 135.5 (C-10a), 140.8
NaHCO solution (200mL), and extracted with ethyl
3
(
(
1
2
CNH -4), 147.5 (CCH -6), 157.0 (CH-3), 161.2
2
acetate (3 · 100mL). The organic layer was washed with
water (3 · 100mL), dried over Na SO , and evaporated
3
COH-1), 162.3 (COH-8), 187.7 (CO-9), 182.0 (CO-
2
4
+
0); HRMS calcd for C H NO : 286.07100, found
86.07109.
at 40ꢂC. The crude product was purified by adsorptive
filtration on silica gel (short column); the first eluate
1
5
12
5
(ethyl acetate) contained traces of emodin and com-
pound 1, the second eluate (methanol) compound 4
5.2.2. 4-[N-(2-Hydroxyethyl)-amino]-emodin (2). Second
isolated product of the reaction mixture of 8. Violet
which was lyophilized to remove solvent residues. Violet
crystals (58mg, 15%); R = 0.3 (methanol) violet, red flu-
crystals (19mg, 12%), mp 225–228ꢂC; R = 0.6 (ethyl
f
f
ꢁ
1
orescence; IR (KBr) cm : 3432, 1577, 1534, 1458, 1394,
acetate/ethanol/water 20:2:1) violet, red fluorescence;
IR (KBr) cm : 3422, 2932, 1585, 1457, 1420, 1383,
ꢁ1
1
1353, 1279; H NMR (methanol-d ) d 2.38 (s, 6H,
4
1
306, 1265, 1206, 1028, 779, 767; H NMR (DMSO-
1
N(CH ) ), 2.44 (s, 3H, CH -6), 2.73 (t, 2H,
3
2
3
3
3
0 0
J = 6.6Hz, CH -2 ), 4.31 (t, 2H, J = 6.6Hz, CH -1 ),
2 2
3
d ) d 2.42 (s, 1H, CH ), 3.56–3.60 (t, 2H, J = 5.1Hz,
6
3
0
3
H-2 ), 3.76–3.80 (t, 2H, J = 5.1, H-1 ), 6.45 (s, 1H, H-
0
5.89 (s, 1H, H-2), 6.86 (s, 1H, H-7), 7.63 (s, 1H, H-5);
C NMR (methanol-d ) d 22.0 (CH -6), 44.4 (CH -
1
3
2
1
), 7.01 (s, 1H, H-7), 7.55 (s, 1H, H-5), 11.31 (br s,
H, OH), 12.39 (s, 1H, OH-8), 13.73 (s, 1H, OH-1);
4 3 2
0
0
2 ), 45.7 (N(CH ) ), 61.4 (CH -1 ), 102.6 (C-9a), 107.9
2
3
2
1
3
0
C NMR (DMSO-d ) d 21.8 (CH -6), 48.4 (CH -2 ),
(CH-2), 109.1 (8a), 117.3 (C-4a), 118.7 (CH-5), 120.4
(C-7), 121.3 (CNH-4), 136.9 (C-10a), 144.6 (CCH -6),
6
3
2
0
61.0 (CH -1 ), 105.5 (C-4a), 108.0 (CH-2), 108.5 (C-
9a), 113.8 (C-8a), 118.9 (CH-7), 120.9 (CH-5), 135.1
2
3
150.8 (COH-3), 162.2 (COH-8), 169.5 (COH-1), 177.1
calcd
(
C-10a), 142.9 (CNH-4), 146.8 (CCH -6), 160.0 (COH-
3
(CO-9),
C H N O : 357.14450, found 357.14523.
180.9
(CO-10);
HRMS
for
+
3
), 161.0 (COH-1), 161.3 (COH-8), 184.9 (CO-9), 179.6
1
9
21
2
5
+
(CO-10); HRMS calcd for C H NO : 330.09722,
17 16 6
found 330.09727.
5.2.5. 4-[N-(3-Aminopropyl)-amino]-emodin (5). (Diacet-
oxyiodo)benzene (354mg, 1.1mmol) was added to a
solution of emodin (270mg, 1.0mmol) in propylenedi-
amine (40mL) and stirred for 16h at room temperature.
The reaction was monitored by TLC. The solution was
poured into a mixture of ice (200g), 10M HCl
5
.2.3. 4-[N-(2-Aminoethyl)-amino]-emodin (3). (Diacet-
oxyiodo)benzene (354mg, 1.1mmol) was added to a
solution of emodin (270mg, 1.0mmol) in ethylenediam-
ine (40mL) and stirred for 5h at room temperature. The
reaction was monitored by TLC. The solution was
poured into a mixture of ice (200g) and 10M HCl
(60mL), neutralized with saturated NaHCO solution
3
(200mL), and extracted with ethyl acetate
(3 · 100mL). The organic layer was washed with water
(3 · 100mL), dried over Na SO , and evaporated at
(
(
60mL), neutralized with saturated NaHCO solution
3
200mL) and extracted with ethyl acetate (3 · 100mL).
2
4
The organic layer was washed in water (3 · 100mL),
dried over Na SO , and evaporated at 40ꢂC. The crude
40ꢂC. The crude product was purified by adsorptive fil-
tration on silica gel (short column), the first eluate (ethyl
2
4

L. Teich et al. / Bioorg. Med. Chem. 12 (2004) 5961–5971
5967
0
0
acetate) contained traces of emodin and compound 1,
the second eluate (methanol) 5. Violet crystals (260mg,
2.66 (s, 2H, H-2 , H-6 ), 6.82 (s, 1H, H-2), 7.08 (s, 1H,
H-7), 7.58 (s, 1H, H-5), 12.10 (s, 1H, OH-8), 13.13 (s,
1
3
7
tate/ethanol/water 20:2:1) violet, red fluorescence; IR
6%), mp 180ꢂC (decomposition); R = 0.2 (ethyl ace-
1H, OH-1); C NMR (CDCl ) d 22.5 (CH -6), 23.9
3 3
f
0 0 0 0 0
(CH -4 ), 27.7 (CH -3 , CH -5 ), 49.1 (CH -2 , CH -6 ),
2 2 2 2 2
ꢁ
1
KBr) cm : 3440, 2924, 1589, 1526, 1420, 1383, 1276,
(
98.6 (C-4a), 106.9 (CH-2), 109.2 (C-9a), 113.4 (C-8a),
120.8 (CH-5), 124.0 (CH-7), 130.3 (C-10a), 134.6
(CNH-4), 148.2 (CCH -6), 162.0 (COH-8), 163.4
(COH-1), 164.6 (COH-3), 183.5 (CO-10), 190.7 (CO-9);
HRMS calcd for C H NO : 354.13360, found
354.13360.
1
1
208, 422; H NMR (methanol-d ) d 2.04 (t, 2H,
4
3
0
0
J = 3.0Hz, CH -2 ), 2.39 (s, 3H, CH -6), 3.16 (t, 2H,
0
2
3
3
3
J = 3.0Hz, CH -3 ), 4.12 (br s, 2H, CH -1 ), 5.96 (s,
2
2
1
3
+
5
1
H, H-2), 6.80 (s, 1H, H-7), 7.46 (s, 1H, H-5).
C
2
0
20
0
NMR (methanol-d ) d 20.9 (CH -6), 28.8 (CH -2 ),
4
3
2
0 0
6.9 (CH -3 ), 40.7 (CH -1 ), 102.5 (C-9a), 107.9 (CH-
2 2
3
2
), 110.1 (C-8a), 115.5 (C-4a), 118.2 (CH-5), 120.3
5.2.8. 4-(N-Morpholinyl)-emodin (8). (Diacetoxy-
iodo)benzene (190mg, 0.6mmol) was added to a solu-
tion of emodin (135mg, 0.5mmol) in morpholine
(25mL) and stirred for 48h at room temperature.
Morpholine was removed in vacuo, and the residue
was extracted with ethyl acetate (3 · 100mL). The or-
ganic layer was washed with 0.05M HCl (150mL) and
water (3 · 100mL). The solvent was evaporated and
the crude product purified by column chromatography
in two steps first on silica gel (petroleum ether/acetone/
ethyl acetate 1:1:1), then on Sephadex LH-20 (metha-
(
CH-7), 120.3 (CNH-4), 135.5 (C-10a), 144.6 (CCH3-
), 148.2 (COH-3), 161.3 (COH-8), (COH-1), 172.4
CO-9), 181.6 (CO-10). HRMS calcd for
C H N O : 343.12885, found 343.12879.
6
(
+
1
8
19
2
5
5
.2.6. 4-(N-Cyclohexyl-amino)-emodin (6). (Diacetoxy-
iodo)benzene (354mg, 1.1mmol) was added to a solu-
tion of emodin (270mg, 1.0mmol) in cyclohexylamine
(
40mL) and stirred for 8days at room temperature.
The reaction was monitored by TLC (ethyl acetate/eth-
anol/water 20:2:1). When the consumption of emodin
was complete, the solution was poured into a mixture
of ice (400g), 10M HCl (40mL), neutralized with satu-
nol). Violet crystals (35mg, 20%), mp 205ꢂC; R = 0.9
f
(ethyl acetate/ethanol/water 20:2:1) yellow-orange, no
fluorescence; IR (KBr) cm : 3423, 3924, 2863, 1676,
ꢁ
1
1
1628, 1490, 1449, 1327, 1228, 1108; H NMR (CDCl )
rated NaHCO solution (200mL), and extracted with
3
3
0
0
ethyl acetate (3 · 100mL). The organic layer was
washed with water (3 · 100mL), dried over Na SO
and evaporated at 40ꢂC. The crude product was purified
by recrystallization from ethanol by adding water drop-
d 2.48 (s, 3H, CH ), 3.80–4.04 (m, 8H, H-2 , H-3 , H-
5 , H-6 ), 6.85 (s, 1H, H-2), 7.10 (s, 1H, H-7), 7.60 (s,
1H, H-5), 9.58 (br s, 1H, OH-3), 12.02 (s, 1H, OH-8),
3
0
0
2
4
1
3
13.10 (s, 1H, OH-1); C NMR (CDCl ) d 22.4 (CH ),
3 3
0 0 0 0
47.8 (CH -3 , CH -5 ), 68.3 (CH -2 , CH -6 ), 107.4
2 2 2 2
wise. Red needles (56mg, 15%), mp 213–228ꢂC; R = 0.7
f
(
(
chloroform/methanol 14:1) violet, red fluorescence; IR
KBr) cm : 3440, 2926, 1630, 1586, 1522, 1457, 1362,
(CH-2), 109.8 (C-9a), 113.2 (C-8a), 121.1 (CH-5),
124.2 (CH-7), 131.1 (COH-4), 132.4, 134.2 (C-4a, C-
10a), 148.6 (CCH -6), 162.0 (COH-3), 162.8 (COH-1),
ꢁ
1
1
1
302, 1263, 1241, 1207; H NMR (acetone-d ) d 1.35
6
3
0
0
0
(
m, 1H, H-4 a), 1.39 (br s, 2H, H-5 b, 3 b), 1.42 (br s,
0
164.3 (COH-8), 183.5 (CO-10), 191.0 (CO-9); HRMS
calcd for C H NO : 356.11268, found 356.11235.
0
0
+
2
H-5 a, 3 a), 2.10 (br s, 2H, H-2 a, 6 a), 2.47 (s, 3H,
H, H-2 b, 6 b), 1.60 (br s, 1H, H-4 b), 1.80 (br s, 2H,
0
1
9
18
6
0
0
0
0
CH -6), 4.47 (br s, 1H, CH-1 ), 6.62 (s, 1H, H-2), 7.07
3
5.3. Synthesis of annelated dihydrooxazole derivatives
1
3
(
s, 1H, H-7), 7.71 (s, 1H, H-5); C NMR (DMSO-d )
6
0
0
0
d 21.7 (CH -6), 23.8 (CH -3 , 5 ), 25.2 (CH -4 ), 34.4
5.3.1. 4,6-Dihydroxy-2-methyl-8a,9,10,11-tetrahydro-8-
oxa-11a-aza-pentaleno[1,2-a]anthracen-5,12-dion
3
2
2
0
0
0
(
CH -2 , 6 ), 52.9 (CH-1 ), 105.4 (C-9a), 108.2 (CH-2),
(9).
2
1
7
1
1
3
08.8 (C-4a), 114.0 (C-8a), 119.0 (CH-5), 121.0 (CH-
), 135.1 (C-10a), 142.1 (CNH-4), 146.9 (CCH -6),
(Diacetoxyiodo)benzene (190mg, 0.6mmol) was added
to a solution of emodin (135mg, 0.5mmol) in pyrrol-
idine (20mL) and stirred for 2days at room temperature.
The reaction was monitored by TLC (ethyl acetate/eth-
anol/water 20:2:1). When the consumption of emodin
was complete, the solution was poured into a mixture
of ice (200g) and 10M HCl (40mL) and extracted with
ethyl acetate (3 · 100mL). The organic layer was
washed with water (3 · 100mL), dried over Na SO
3
61.0 (COH), 161.0 (COH), 161.4 (COH), 179.9 (CO-
0), 184.9 (CO-9); HRMS calcd for C H NO :
68.14925, found 368.14908.
+
2
1
18
5
5
.2.7. 4-(N-Piperidinyl)-emodin (7). (Diacetoxyiodo)benz-
ene (190mg, 0.6mmol) was added to a solution of emo-
din (135mg, 0.5mmol) in piperidine (20mL) and stirred
for about 8h at room temperature. The solution was
poured into a mixture of ice (200g), 10M HCl (30mL),
and extracted with ethyl acetate (3 · 100mL). The organ-
ic layer was washed with water (3 · 100mL), dried over
Na SO , and evaporated at 20ꢂC. The crude product
2
4
and evaporated at 40ꢂC. The crude product was purified
by column chromatography on silica gel (ethyl acetate).
Violet crystals (30mg, 18%), mp 148–158ꢂC; R = 0.9
f
(ethyl acetate/ethanol/water 20:2:1) pink, orange fluores-
cence; IR (KBr) cm : 2920, 1619, 1851, 1619, 1586,
ꢁ
1
2
4
was purified by column chromatography in two steps
first on silica gel (petroleum ether/acetone/ethyl acetate
1481, 1429, 1358, 1284, 1248, 1215, 1176, 1057, 777;
H NMR (CDCl ) 1.89 (m, 1H, H-10a), 1.96 (m, 1H,
1
3
1
:1:1), then on Sephadex LH-20 (methanol). Orange-
H-10b), 1.98 (m, 1H, H-9a), 2.49 (s, 3H, CH ), 2.49
3
brown crystals (27mg, 15%), mp 140ꢂC (decomposition);
(br s, 1H, H-9b), 3.01 (m, 1H, H-11a), 6.06 (br s, 1H,
H-8a), 6.46 (s, 1H, H-7), 7.08 (s, 1H, H-3), 7.65 (s,
1H, H-1), 12.39 (s, 1H, OH-4), 13.52 (s, 1H, OH-6);
R = 0.8 (ethyl acetate/ethanol/water 20:2:1) yellow, no
f
ꢁ
1
fluorescence; IR (KBr) cm : 3441, 2934, 1560, 1498,
1
1
13
412, 1363, 1283, 1260; H NMR (CDCl ) d 1.60–1.84
C NMR (CDCl ) d 22.3 (CH -2), 23.8 (CH -10),
3
3
3
2
0
0
0
(
m, 6H, H-3 , H-4 , H-5 ), 2.48 (s, 3H, CH -6), 2.63–
32.6 (CH -9), 56.4 (CH -11), 102.1 (CH-7), 105.4
2 2
3

5968
L. Teich et al. / Bioorg. Med. Chem. 12 (2004) 5961–5971
(
CH-8a), 107.8 (C-5a), 108.5 (CO-7a), 114.4 (C-4a),
(C-12b), 162.1 (COH-8), 163.4 (COH-6), 181.6 (CO-
12), 183.4 (CO-7). HRMS calcd for C H N O :
311.10263, found 311.10298.
+
1
1
1
20.4 (CH-1), 123.6 (CH-3), 130.4 (C-11c), 134.2 (C-
2a), 137.6 (C-11b), 147.9 (CH-2), 162.3 (COH-4),
64.8 (COH-6), 180.8 (CO-12), 189.2 (CO-5); HRMS
1
7
15
2
4
+
calcd for C H NO : 338.10230, found 338.10239.
1
5.4.2. 7,9-Dihydroxy-11-methyl-1,2,3,4,4a,5,14,14a-octa-
hydro-naphto[2,3-a]phenazine-8,13-dione (12). (Diacet-
oxyiodo)benzene (354mg, 1.1mmol) was added to a
solution of emodin (270mg, 1.0mmol) in (+/ꢁ)trans-
9
17
5
5
.3.2. 4,6-Dihydroxy-2-methyl-9,10,11,12-tetrahydro-
aH-8-oxa-12a-aza-indeno[1,2-a]-anthracen-5,13-dione (10).
8
Second isolated product of the reaction mixture leading
to 4-(N-piperidinyl)-emodin 7. Violet crystals (28mg,
1
water 20:2:1) violet, red fluorescence; IR (KBr) cm
1
,2-diaminocyclohexane (40mL) and stirred for 7days
at room temperature. The reaction was monitored by
TLC. The solution was poured into a mixture of ice
6%), mp 118–120ꢂC; R = 0.9 (ethyl acetate/ethanol/
f
(200g), 10M HCl (60mL), neutralized with saturated
NaHCO solution (200mL), and extracted with ethyl
ꢁ
1
:
441, 2937, 2857, 1618, 1492, 1445, 1364, 1293, 1255,
214, 833, 770; H NMR (CDCl ) d 1.65–1.84 (m, 4H,
3
3
1
acetate (3 · 100mL). The organic layer was washed with
water (3 · 100mL), dried over Na SO , and evaporated
1
3
2
4
H-10, H-11), 2.34 (m, 2H, H-9), 2.48 (s, 3H, CH ),
3
H-12, J = 10.2Hz), 5.66 (d, 1H, H-8a, J = 9.3Hz),
6
1
(
3
1
(
3
at 40ꢂC. The crude product was purified by column
chromatography on silica gel (ethyl acetate). Violet crys-
3
.27–3.34 (t, 1H, H-12, J = 12Hz), 5.03–5.07 (d, 1H,
3
3
tals (218mg, 60%), mp 145–155ꢂC; R = 0.8 (ethyl ace-
f
.44 (s, 1H, H-7), 7.03 (s, 1H, H-3), 7.57 (s, 1H, H-1),
2.40 (s, 1H, OH-4), 14.16 (s, 1H, OH-6); C NMR
tate/ethanol/water 20:2:1) violet, pink fluorescence; IR
(KBr) cm : 3422, 2929, 2858, 1586, 1513, 1355, 1311,
1
3
ꢁ1
CDCl ) d 22.4 (CH ), 25.3 (CH -10), 30.0 (CH -11),
1
3
3
2
2
1
1
1
2
270, 1225, 1196, 1178, 636; H NMR (DMSO-d ) d
6
2.6 (CH -9), 48.4 (CH -12), 98.5 (CH-8a), 100.3 (C-
0
0
2
2
.23–1.40 (m, 2H, H-1 , 4 ), 1.39 (br s, 2H, H-2b, 3b),
00
2c), 102.0 (CH-7), 107.4 (C-5a), 114.4 (C-4a), 119.9
00
.78 (br s, 2H, H-2a, 3a), 2.02 (br s, 2H, H-1 , 4 ),
.40 (s, 3H, CH -11), 3.08 (br s, 2H, H-14a, 4a), 6.10
CH-1), 122.5 (CH-3), 135.4 (C-13a), 137.3 (C-12b),
47.4 (CCH -2), 162.0 (COH-4), 162.0 (C-7a), 164.2
3
1
3
(s, 1H, H-6), 6.97 (s, 1H, H-10), 7.45 (s, 1H, H-12),
(COH-6), 181.1 (CO-13), 187.8 (CO-5); HRMS calcd
for C H NO : 352.1185, found 352.1182.
8
1
.14 (s, 1H, NH-5), 10.41 (s, 1H, NH-14), 12.72 (s,
H, OH-9), 13.80 (s, 1H, OH-7); C NMR (DMSO-
+
13
2
0
18
5
d ) d 21.7 (CH -11), 23.5, 23.6 (CH -2, 3), 29.5, 29.6
6
3
2
5
.4. Synthesis of annelated tetrahydropyrazine derivatives
(CH -1, 4), 53.6, 53.8 (CH-14a, 4a), 100.6 (CH-6),
102.8 (C-7a), 106.4 (C-13a), 114.5 (C-8a), 118.5 (CH-
12), 121.2 (CH-10), 134.5 (C-12a), 137.7 (C-13b), 145.8
2
5
4
.4.1. 6,8-Dihydroxy-10-methyl-1,2,3,4-tetrahydro-1,
-diaza-benzo[a]anthracen-7,12-dione (11). (Diacetoxy-
(CCH -11), 147.5 (C-5a), 160.9, 162.2 (COH-7, 9),
3
iodo)benzene (180mg, 0.56mmol) was added to a solu-
tion of emodin (135mg, 0.5mmol) in ethylenediamine
180.8 (CO-13), 182.3 (CO-8); HRMS calcd for
C H N O : 365.14958, found 365.14978.
+
2
1
21
2
4
(
20mL) and stirred for 4days at room temperature.
The reaction was monitored by TLC (ethyl acetate/eth-
anol/water 20:2:1). Once the consumption of emodin
was complete, the solution was poured into a mixture
of ice (200g) and 10M HCl (40mL), neutralized with
saturated NaHCO3 solution (200mL), and extracted
with ethyl acetate (3 · 100mL). The organic layer was
washed with water (3 · 100mL), dried over Na SO ,
5.4.3. 6,8-Dihydroxy-4,10-dimethyl-1,2,3,4-tetrahydro-
1,4-diaza-benzo[a]anthracen-7,12-dione (13). (Diacet-
oxyiodo)benzene (180mg, 0.56mmol) was added to a
solution of emodin (135mg, 0.5mmol) in N-methylpip-
erazine (20mL) and stirred for 9days at room tempera-
ture. The reaction was monitored by TLC (ethyl acetate/
ethanol/water 20:2:1). Once the consumption of emodin
was complete, the solution was poured into a mixture of
ice (200g) and 10M HCl (40mL), neutralized with satu-
2
4
and evaporated at 40ꢂC. The crude product was purified
by column chromatography on silica gel (ethyl acetate).
To remove solvent residues, the product was dissolved in
methanol (30mL), and water (50mL) was added fol-
lowed by evaporation at 40ꢂC and lyophilization. Black
rated NaHCO solution (200mL), and extracted with
3
ethyl acetate (3 · 100mL). The organic layer was
washed with water (3 · 100mL), dried over Na SO ,
2
4
powder (160mg, 45%), mp 271–276ꢂC; R = 0.8 (ethyl
and evaporated at 40ꢂC. The crude product was purified
by column chromatography on silica gel (ethyl acetate).
Dark violet powder (160mg, 60%), mp 200ꢂC (decom-
f
acetate/ethanol/water 20:2:1) violet, red fluorescence;
IR (KBr) cm : 3372, 1586, 1526, 1475, 1390, 1347,
ꢁ1
1
1
327, 1274, 1199, 1175; H NMR (CDCl ) d 2.37 (s,
position); R = 0.6 (ethyl acetate/ethanol/water 20:2:1)
3
f
ꢁ1
3
CH -2), 4.97 (br s, 1H, NH-4), 6.18 (s, 1H, H-5), 7.02
H, CH -10), 3.62 (br s, 2H, CH -3), 3.70 (br s, 2H,
violet, red fluorescence; IR (KBr) cm : 3440, 2924,
1586, 1529, 1474, 1414, 1383, 1346, 1330, 1314, 1284,
3
2
2
1
(
1
s, 1H, H-9), 7.67 (s, 1H, H-11), 10.55 (br s, 1H, NH-
), 12.74 (s, 1H, OH-9), 13.81 (s, 1H, OH-6);
1260, 1182; H NMR (CDCl ) d 2.46 (s, 3H, CH -10),
3
3
1
H
3.04 (s, 3H, CH -4), 3.51 (s, 2H, CH -3), 3.68 (s, 2H,
CH -2), 6.00 (s, 1H, H-5), 6.95 (s, 1H, H-9), 7.79 (s,
3 2
NMR (DMF-d ) d 2.44 (s, 3H, CH -10), 3.60 (s, 2H,
7
3
2
CH -3), 3.70 (s, 2H, CH -2), 6.23 (s, 1H, H-5), 7.00 (s,
1H, H-11), 10.94 (br s, 1H, NH-1), 12.76 (s, 1H, OH-
2
2
1
3
1
1
1
3
H, H-9), 7.59 (s, 1H, H-11), 8.18 (s, 1H, NH-4),
0.73 (s, 1H, NH-1), 12.84 (s, 1H, OH-8), 13.96 (s,
H, OH-6); C NMR (DMF-d ) 22.9 (CH -10), 39.2,
8), 13.88 (s, 1H, OH-6); C NMR (CDCl ) d 22.5
3
(CH -10), 39.2 (CH -4), 40.1 (CH -2), 48.2 (CH -3),
3
3
2
2
1
3
101.4 (CH-5), 104.3 (C-6a), 107.1 (C-12a), 115.2 (C-
7a), 119.2 (CH-11), 121.7 (CH-9), 135.4 (C-11a), 138.8
(C-12b), 146.0 (CCH -10), 146.2 (CN-4a), 161.9
7
3
9.4 (CH -3, CH -4), 101.3 (CH-5), 103.8 (C-6a), 107.4
2
2
(
1
C-12a), 115.5 (C-7a), 119.05 (CH-11), 121.6 (CH-9),
35.7 (C-11a), 138.7 (C-4a), 146.5 (CCH -10), 148.3
3
(COH-6), 162.1 (COH-8), 182.2 (CO-12), 184.9
3

L. Teich et al. / Bioorg. Med. Chem. 12 (2004) 5961–5971
5969
+
CO-7); HRMS calcd for C H N O : 325.11828,
ꢁ1
(
found 325.11928.
water 20:2:1) violet, red fluorescence; IR (KBr) cm
3408, 3110, 2950, 1700, 1583, 1527, 1457, 1420, 1380,
:
1
8
17
2
4
1
1
261, 1205, 779; H NMR (DMSO-d ) d 1.88 (m, 2H,
6
0
CH -2 ), 2.43 (s, 3H, CH -6), 3.34 (dt, 2H, J = 6.0Hz,
2 3
3
5
5
.5. Synthesis of amides of compound 5
.5.1. Amide (14). Acetic anhydride (46lL, 0.5mmol)
3
0
0
J = 6.0Hz, CH -3 ), 3.92 (br s, 2H, CH -1 ), 6.42 (s,
2
2
1H, H-2), 7.00 (s, 1H, H-7), 7.55 (s, 1H, H-5), 7.43–
0
0
0
0
3
7.80 (m, 4H, H-3 , 4 , 5 , 6 ), 8.41 (t, 1H, J = 6.0Hz,
was added to a solution of 5 (170mg, 0.5mmol) in dry
methanol (15mL) and stirred at room temperature for
0
CONH), 11.34 (br s, 1H, NH-1 ), 12.44 (s, 1H, OH-8),
13.84 (s, 1H, OH-1);
1
3
2
h. Water (20mL) was added and methanol was evapo-
C NMR (DMSO-d ) 21.8
6
0
0
0
rated in vacuo. The violet precipitate was filtered off and
dried in vacuo. The crude product was purified by col-
umn chromatography on silica gel (ethyl acetate). Dark
(CH -6), 30.9 (CH -2 ), 36.7 (CH -3 ), 43.5 (CH -1 ),
3
2
2
2
105.2 (C-9a), 107.9 (C-4a), 108.4 (CH-2), 113.9 (C-8a),
118.6 (CH-5), 120.8 (CH-7), 127.6 (CH-3 ), 129.0
00
00 00 00 00
violet powder (86mg, 45%), mp 183–194ꢂC; R = 0.5
(CH-5 ), 129.2 (CH-6 ), 130.5 (C-1 ), 131.2 (CH-4 ),
135.0 (C-10a), 138.0 (C-2 ), 143.1 (C-4), 146.6 (CCH -
f
00
(
cence; IR (KBr) cm : 3384, 3110, 2955, 1636, 1584,
ethyl acetate/ethanol/water 20:2:1) violet, red fluores-
3
ꢁ
1
6), 160.6 (COH-3), 161.0 (COH-1), 161.6 (COH-8),
167.9 (COOH), 168.6 (CONH), 179.6 (CO-10), 184.3
(CO-9); HRMS calcd for C H N O : 491.14489,
found 491.14502.
5.6. Synthesis of tetranitro-emodin derivatives
5.6.1. 1,8-Dihydroxy-2,4,5,7-tetranitro-anthraquinone
1
527, 1458, 1340, 1508, 1261, 1206; H NMR (pyr-
1
3
+
idine-d ) d 2.05 (s, 3H, Ac), 2.10 (m, 2H, J = 7Hz,
5
26 23
2
8
0
3
CH -2 ), 2.27 (s, 3H, CH -6), 3.65 (dt, 2H, J = 6Hz,
2
3
3
0
3
J = 7Hz, CH -3 ), 4.21 (t, 2H, J = 7Hz, CH -1 ),
0
2
2
6
8
.69 (s, 1H, H-2), 7.08 (s, 1H, H-7), 7.99 (s, 1H, H-5),
.72 (br s, 1H, NHAc); C NMR: due to the very low
1
3
solubility in suitable solvents, the need for long record-
ing times, and the instability of compound 14, no satis-
(17). Concentrated HNO (4mL) was added to a solu-
3
tion of 1,8-dihydroxy-anthraquinone (240mg, 1.0mmol)
in concentrated H SO (5mL) (CAUTION, ice bath).
1
factory C NMR could be obtained; HRMS calcd for
3
+
C H N O : 385.13996, found 385.13963.
2
0
21
2
6
2
4
After 2h stirring at room temperature the mixture was
poured into ice (100g). The yellow precipitate was fil-
tered off, washed with water (20mL), and dried in
vacuo. The crude product was purified by adsorptive fil-
tration on silica gel (short column, methanol). Yellow
powder (310mg, 74%), mp > 200ꢂC (decomposition);
5
0
0
.5.2. Amide (15). Benzoic acid anhydride (113mg,
.5mmol) was added to a solution of 5 (170mg,
.5mmol) in dry methanol (15mL) and stirred at room
temperature for 2h. Water (20mL) was added and
methanol was evaporated in vacuo. The violet precipi-
tate was filtered off and dried in vacuo. The crude prod-
uct was purified by column chromatography on silica gel
R = 0.2 (ethyl acetate/ethanol/water 20:2:1) red, no fluo-
f
ꢁ
1
rescence; IR (KBr) cm : 3075, 1639, 1587, 1418, 1370,
1
1252, 1106, 716; H NMR (DMSO-d ) 8.66 (s, 2H, H-3
(
ethyl acetate). Dark violet powder (100mg, 45%), mp
6
1
3
1
2
2
1
91–200ꢂC; R = 0.6 (ethyl acetate/ethanol/water
and H-6); C NMR (DMSO-d ) d 121.2 (C-8a, C-9a),
f
6
ꢁ
1
0:2:1) violet, red fluorescence; IR (KBr) cm : 3424,
932, 1636, 1586, 1526, 1490, 1458, 1420, 1358, 1259,
203; H NMR (DMSO-d ) d 1.91 (m, 2H, CH -2 ),
127.0 (C-3, C-6), 133.4 (C-4, C-5), 134.8 (C-4a, C-10a),
143.1 (C-2, C-7), 160.1 (C-1, C-8), 181.4 (C-9), 184.9
1
0
ꢁ
(C-10); HRMS calcd for C H N O : 418.97530,
14
6
2
3
4
12
3
.44 (s, 3H, CH -6), 3.34 (dt, 2H, J = 6.0Hz,
2
found 418.97481.
3
3
0
0
J = 6.0Hz, CH -3 ), 3.92 (br s, 2H, CH -1 ), 6.45 (s,
2
2
0
1
4
2
1
H, H-2), 7.04 (s, 1H, H-7), 7.34–7.53 (m, 3H, H-3 ,
5.6.2. 1,3,8-Trihydroxy-6-methyl-2,4,5,7-tetranitro-
0
0
0
0
3
, 5 ), 7.58 (s, 1H, H-5), 7.86 (d, 2H, J = 7.0Hz, H-
anthraquinone (18). Concentrated HNO3 (4mL) was
added dropwise to a solution of emodin (270mg,
1.0mmol) in concentrated H SO (5mL) (CAUTION,
3
, 6 ), 8.56 (t, 1H, J = 6.0Hz, CONH), 11.34 (br s,
0
H, NH-1 ), 12.45 (s, 1H, OH-8), 13.83 (s, 1H, OH-1);
2
4
1
3
0
C NMR (DMSO-d ) d 21.7 (CH -6), 31.1 (CH -2 ),
ice bath). After 2h of stirring at room temperature,
the mixture was poured into ice (100g) and extracted
with ethyl acetate (3 · 100mL). The organic layer was
washed with water (3 · 100mL), dried over Na SO ,
6
3
2
0 0
7.0 (CH -3 ), 43.7 (CH -1 ), 105.6 (C-9a), 108.2 (CH-
2 2
3
2
), 108.5 (C-8a), 113.9 (C-4a), 118.9 (CH-5), 121.0
00 00 00 00
CH-7), 127.1 (CH-2 , 6 ), 128.2 (CH-3 , 5 ), 131.0
(
(
2
4
00
00
CH-4 ), 134.5 (C-1 ), 135.1 (C-10a), 143.1 (C-4),
46.7 (CCH -6), 161.0 (COH-3), 161.5 (COH-1), 161.5
COH-8), 166.2 (CONH), 179.8 (CO-10), 184.7 (CO-
and evaporated. The crude product was purified by
adsorptive filtration on silica gel (short column, ethyl
acetate) and lyophilized. Red powder (300mg, 67%),
1
(
3
+
9
); HRMS calcd for C H N O : 447.15506, found
2
mp > 200ꢂC decomposition; R = 0.4 (ethyl acetate/eth-
5
23
2
6
f
4
47.15497.
anol/water 20:2:1) red, no fluorescence; IR (KBr)
H
ꢁ
1
cm : 3432, 1618, 1534, 1420, 1376, 1248, 1186;
1
1
3
5
0
(
.5.3. Amide (16). Phthalic acid anhydride (74mg,
.5mmol) was added to a solution of compound 5
170mg, 0.5mmol) in dry methanol (15mL) and stirred
NMR (methanol-d ) d 2.08 (s, 3H, 6-CH ), C NMR
4 3
(methanol-d ). Due to the lack of protons, even pro-
longed recording times show only the methyl carbon: d
4
ꢁ
at room temperature for 2h. Water (20mL) was added
and methanol was evaporated in vacuo. The violet pre-
cipitate was filtered off and dried in vacuo. The crude
product was purified by column chromatography on sil-
ica gel (ethyl acetate). Dark violet powder (196mg,
11.4 (6-CH3); HRMS calcd for C H N O
15
:
5
4
13
448.98532, found 448.98341.
5.6.3. 1,8-Diacetyl-3-hydroxy-6-methyl-2,4,5,7-tetranitro-
anthraquinone (19). Some drops of concentrated
H SO were added to a solution of 18 (113mg,
8
0%), mp 155–165ꢂC; R = 0.3 (ethyl acetate/ethanol/
f
2
4

5970
L. Teich et al. / Bioorg. Med. Chem. 12 (2004) 5961–5971
0
.25mmol) in acetic anhydride (15mL). The solution
pound and 10lM doxorubicin in serum-free DME med-
ium for 24h. After two washing steps in ice cold
phosphate buffered saline cells were lysed in 5mM
Tris–HCl by sonification for 5s. Fluorescence of doxo-
rubicin was measured at 590nm upon excitation at
485nm with Mithras LB 940 multi-label reader (Bert-
hold Technologies, Bad Wildbach, Germany). Values
were corrected for the intrinsic fluorescence of the deriv-
atives determined by incubating cells under same condi-
tions but without doxorubicin and expressed as
percentage of the enhancement of doxorubicin uptake
produced by 2lM cyclosporine A.
was stirred at room temperature for two weeks, poured
into ice (100g), and extracted with ethyl acetate
(
(
3 · 100mL). The organic layer was washed with water
3 · 100mL), dried over Na SO , and evaporated. The
2
4
crude product was purified by adsorptive filtration on
silica gel (short column, ethyl acetate). Orange powder
(34mg, 25%), mp > 200ꢂC decomposition; R = 0.4
(ethyl acetate/ethanol/water 20:2:1) orange-yellow, no
f
ꢁ
1
fluorescence; IR (KBr) cm : 3440, 1794, 1696, 1636,
1
d ) d 2.36 (s, 3H, CH CO), 2.38 (s, 3H, CH CO), 2.47
1
598, 1548, 1367, 1274, 1245, 1175; H NMR (DMF-
7
3
3
1
3
(
s, 3H, CH -6); C NMR (DMF-d ) d 12.8 (CH -6),
3 7 3
2
calcd for C H N O : 533.00699, found 533.00640.
0.7, 20.9 (CH CO), 168.0, 168.2 (CH CO); HRMS
3 3
ꢁ
Acknowledgements
1
9
9
4
15
The authors gratefully acknowledge the Deutsche Fors-
chungsgemeinschaft (DFG) Graduiertenkolleg ÔMecha-
nisms and Applications of Non-Conventional Oxidation
ReactionsÕ for their academic (Katja S. Daub, Kurt Eger
and Lars Teich) and financial support. Parts of this
work were supported by a grant from the Deutsche Fors-
chungsgemeinschaft (Ge 465/7-1). K.E. thanks the Fonds
der Chemischen Industrie for financial support. We
thank Professor Joachim Sieler for generating X-ray
crystallographic data from 13 and for technical support.
The excellent technical assistance of J. Ortwein, D. Kel-
lert and F. Struck is gratefully acknowledged.
6
. Biological assays
Primary rat hepatocytes and HepG2 hepatoblastoma
cells were used for studying cytotoxicity and inhibition
of proliferation. Primary hepatocytes were isolated and
cultured in serum-free modified Williams medium E as
described previously. Test compounds were added
after 2h of cultivation and the cytotoxic response was
2
0
2
1
determined after 24h using the MTT assay. HepG2
8,22
1
cells were maintained as described
and incubated
with the test compounds similarly as primary cells for
measuring cytotoxicity. EC50 values were determined
by curve fitting of concentration versus viability plots
(
cf. Fig. 8), and are given as means ± SD. Where solubil-
References and notes
ity of the respective compound was limited, EC50 values
particularly in the upper range could not be determined
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1
4
3, 1550.
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1
5. Chrysophanol [1,8-dihydroxy-3-methyl-anthraquinone],
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2
5
H4-II-E similarly as described. Briefly, 0.6 million cells
were cultured in DME medium supplemented with 10%
FCS for 24h and the coincubated with 40lM test com-

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2