Synthesis and biological evaluation of novel 10-benzyl-substituted 4,5-dichloro-10H-anthracen-9-ones as inhibitors of keratinocyte hyperproliferation

Article abstract of DOI:10.1016/j.ejmech.2010.08.047

A series of 10-substituted 4,5-dichloro-10H-anthracen-9-ones were synthesized in the search for novel agents against keratinocyte hyperproliferation. The antiproliferative activity of these novel anthrones was evaluated using the human keratinocyte line HaCaT as the primary test system. Structure-activity relationships with respect to the nature and position of the substituents at the benzyl moiety were studied, with a 3-hydroxy-4-methoxy- substitution pattern being the most potent (IC₅₀ = 0.7 μM) and comparable to the potency of the antipsoriatic anthralin. In contrast to anthralin, inhibition of keratinocyte hyperproliferation was not mediated by damage to the keratinocyte membrane, as the activity of lactate dehydrogenase released from the cytoplasm was in the control range. These findings may be rationalized as a benefit of the ineffectiveness of the novel anthrones to interact with the free radical 2,2-diphenyl-1-picrylhydrazyl.

Full text of DOI:10.1016/j.ejmech.2010.08.047

European Journal of Medicinal Chemistry 45 (2010) 5278e5285
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel 10-benzyl-substituted
4,5-dichloro-10H-anthracen-9-ones as inhibitors of keratinocyte
hyperproliferation
*
Ulrich Kratz, Helge Prinz, Klaus Müller
Institute of Pharmaceutical and Medicinal Chemistry, Westphalian Wilhelms-University, Hittorfstraße 58e62, D-48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 10-substituted 4,5-dichloro-10H-anthracen-9-ones were synthesized in the search for novel
agents against keratinocyte hyperproliferation. The antiproliferative activity of these novel anthrones
was evaluated using the human keratinocyte line HaCaT as the primary test system. Structureeactivity
relationships with respect to the nature and position of the substituents at the benzyl moiety were
Received 16 June 2010
Received in revised form
17 August 2010
Accepted 22 August 2010
Available online 9 September 2010
studied, with a 3-hydroxy-4-methoxy-substitution pattern being the most potent (IC50 ¼ 0.7
mM) and
comparable to the potency of the antipsoriatic anthralin. In contrast to anthralin, inhibition of kerati-
nocyte hyperproliferation was not mediated by damage to the keratinocyte membrane, as the activity of
lactate dehydrogenase released from the cytoplasm was in the control range. These findings may be
rationalized as a benefit of the ineffectiveness of the novel anthrones to interact with the free radical 2,
2-diphenyl-1-picrylhydrazyl.
Keywords:
Anthrone
Antiproliferative activity
Antipsoriatic
HaCaT cells
Ó 2010 Elsevier Masson SAS. All rights reserved.
Radical
1. Introduction
A former strategy to provide agents with diminished intensity of
oxygen-radical generation was to keep the 1,8-dihydroxy substi-
First line management of psoriasis, a chronic relapsing, hyper-
proliferative skin disease [1], is with topical treatments, including
topical corticosteroids, tar-based preparations, and anthralin [2].
While anthralin (dithranol, 1,8-dihydroxy-10H-anthracen-9-one, 1)
used to be a mainstay for the topical treatment of psoriasis, its use
has declined in recent years because of the availability of more
cosmetically acceptable alternatives such as vitamin D analogues
and retinoids [3]. However, these drugs serve only to abate this
hyperproliferative skin disease, whereas antipsoriatic anthrones
such as anthralin clear the pathological features of psoriasis totally
[4,5]. Like most forms of current therapy of psoriasis the benefit of
this drug is limited by undesirable proinflammatory effects on the
skin as well as staining of the skin and clothing [6], which are
thought to be mediated by oxygen radicals [7] and anthralin
metabolite radicals [8,9]. Therefore, the development of a topically
active analogue that obviates these drawbacks is highly desirable.
tution pattern of anthralin unchanged, but to modify the critical
10-position of the molecule [10,11]. The methylene moiety at C-10
is responsible for the production of superoxide radical, a precursor
in the formation of the highly reactive hydroxyl radical by anthralin
[12]. Because of the strong intramolecular hydrogen bonding
between both hydroxy functions and the keto group, the molecule
is relatively stable in the solid state, where it exists exclusively as
the anthrone tautomer. However, in the presence of protic solvents,
basic additives or metal ions, this bonding is disturbed, and ioni-
zation results in the formation of the resonance-stabilized
anthralin anion [7]. This is the key intermediate in the auto-
oxidation process, which easily undergoes one-electron transfer to
oxidizing agents and produces the anthralin free radical. The radical
can dimerize to the inactive bianthrone 2 (Chart 1) or finally
polymerize to the so-called anthralin-brown, which is responsible
for the darker shades of brown staining which occur on the skin
and clothes [13]. Hence, in our ongoing search for anthrone deriv-
atives with an improved benefit/risk ratio, we became interested in
analogues lacking the hydroxy groups.
In this study, we have replaced the 1,8-dihydroxy by
a 4,5-dichloro-substitution pattern. A previous investigation into
the potential of several isomers of anthralin with replaced hydroxy
groups has shown that in contrast to the 1,8-dichloro only the
Abbreviations: DPPH, 2,2-diphenyl-1-picrylhydrazyl; LDH, lactate dehydroge-
nase; RT, room temperature; TEBAC, triethylbenzylammonium chloride.
* Corresponding author. Tel.: þ49 251 833 3324; fax: þ49 251 833 2144.
E-mail address: kmuller@uni-muenster.de (K. Müller).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.047

U. Kratz et al. / European Journal of Medicinal Chemistry 45 (2010) 5278e5285
5279
Chart 1. Structures of anthralin (1) and its corresponding dimer 2.
Scheme 2. Reagents: (a) 4,5-dichloroanthrone, CH2Cl2, NaOH, TEBAC, RT; (b) PdeC, H2,
THF, RT, atmospheric pressure. R is defined in Table 1.
4,5-dichloro-substituted anthrone 3 (Scheme 1) was able to arrest
keratinocyte growth in the low micromolar concentration range
[14]. However, this compound itself was not stable under culture
conditions (see Section 2.1). Therefore, it was deemed necessary to
stabilize the structure with a 10-substituent. While studies on the
evaluation of antiproliferative properties of hydroxy-substituted
anthraquinones and anthrones are vast, there are only few reports
on cell growth inhibition by chloro-substituted anthrones or
anthrone-derived analogues [15e18].
In order to explore structureeactivity relationships of this chem-
ical series of anthrones, we prepared a number of 10-substituted
analogues of 4,5-dichloro-9-anthrone with different substituents at
the terminal phenyl ring of the side chain. We have tested these novel
compounds as inhibitors of keratinocyte hyperproliferation, which is
one of the hallmarks of psoriasis. We also evaluated their capability to
interact with a stable free radical by use of 2,2-diphenyl-1-picrylhy-
drazyl (DPPH). Furthermore, release of lactate dehydrogenase (LDH)
was measured to exclude cytotoxic effects mediated by membrane
damage.
2.2. Synthesis of 4,5-dichloro-9-anthrones
Parent 4,5-dichloro-9-anthrone (3) was prepared from the cor-
responding anthraquinone with sodium dithionite [20], which
selectively reduces the carbonyl group flanked by the peri
substituents of the anthraquinone. The 10-substituted analogues
were synthesized by the methods outlined in Schemes 2 and 3.
Due to the weakly acid nature of C-10 of the anthrone nucleus,
C-10 alkylation with alkyl halides is difficult. However, it can be
accomplished in the presence of a strong base such as sodium
hydride under anhydrous conditions. The reaction of parent
anthrone 3 with benzyl bromide in tetrahydrofuran under these
conditions gave the respective 10-benzyl acridone 5a in a very low
yield. To overcome this drawback, introduction of the 10-benzyl
substituents onto the anthrone nucleus was carried out in the
presence of a phase transfer catalyst (PTC). Accordingly, a two-
phase system consisting of an organic solvent (CH2Cl2) and 30%
aqueous sodium hydroxide solution in the presence of trie-
thylbenzylammonium chloride (TEBAC) was used to synthesize
10-benzylated anthrones in better yields. In this system, TEBAC
transports the deprotonated anthrone from the aqueous to the
organic phase where the benzylation takes place. Scheme 2 pres-
ents the reaction of 3 with the appropriate benzyl chlorides, which
gave exclusively the monobenzylated anthrones 5aet. Products of
C-10 dibenzylation or O-benzylation could not be detected.
Because ether cleavage of 4-methoxy analogue 5h with boron
tribromide, aluminum chloride or hydrobromic acid was not
successful or resulted in total loss of the benzyl moiety at C-10, the
desired phenolic analogues 5uex were prepared by hydrogenolytic
cleavage of benzyl ethers 5qet.
2. Chemistry
2.1. Stability of 4,5-dichoro-9-anthrone
Anthralin is readily oxidized to the corresponding anthraqui-
none and dimer 2 [19]. In order to study the stability of 4,5-
dichloro-9-anthrone (3), reversed phase high-performance liquid
chromatography was employed in the present work to determine
its potential degradation in DMSO solution and in keratinocyte
culture medium under culture conditions at 37 ꢀC. After 48 h of
incubation, anthrone 3 was completely degraded to the corre-
sponding anthraquinone (1,8-dichloroanthracene-9,10-dione) and
its dimer 4, 1,10,8,80-tetrachloro-[9,90-bianthracene]-10,100(9H,90H)-
dione, in DMSO as well as in aqueous culture medium. HPLC
chromatograms of the samples obtained after incubation were
compared with those obtained from samples spiked with authentic
anthraquinone and dimer. Dimer 4 was independently prepared
from anthrone 3 by oxidation with iron(III) chloride (Scheme 1).
For comparison, also some 10-phenyl substituted analogues of 3
were prepared (Scheme 3). The pertinent compounds 7aec were
obtained in
a FriedeleCrafts-type reaction of 10-bromo-4,5-
dichloro-9-anthrone (6) [21] in a suspension of the appropriate
benzene derivative using aluminum chloride as a catalyst.
Scheme 3. Reagents: (a) benzene, toluene, or anisole, AlCl3, reflux. R is defined in
Table 1.
Scheme 1. Reagents: (a) FeCl3, HOAc, N2, reflux.

5280
U. Kratz et al. / European Journal of Medicinal Chemistry 45 (2010) 5278e5285
Table 1
identifying new topical antipsoriatic agents, and it was described as
an extremely sensitive target for the antiproliferative action of the
antipsoriatic drug anthralin [24]. Accordingly, we evaluated the
effects of the novel 4,5-dichloro-9-anthrones on the growth of
cultured HaCaT cells, which was determined directly by counting
the dispersed cells under a phase-contrast microscope after 48 h of
treatment.
Antiproliferative activity against HaCaT keratinocytes, lactate dehydrogenase
release, and free radical interacting capability of 10-substituted 4,5-dichloro-10H-
anthracen-9-ones.
O
O
Anthralin has been reported to cause membrane damage, but
did not directly lead to substantial membrane destruction [24]. In
order to confirm that inhibition of keratinocyte hyperproliferation
by the most potent compounds is not a result of non-specific toxic
effects against keratinocyte cell membranes by causing leakage of
cytoplasm through it, plasma membrane integrity was tested by the
release of lactate dehydrogenase (LDH) into the culture medium.
Furthermore, free radical production by anthralin and the
resulting dimerization and polymerization of the molecule is
associated with the instability of the drug as well as with unde-
sired side effects such as staining and irritation of the skin [7]. In
order to compare the capability of the novel 10-substituted 4,5-
dichloro-9-anthrones to produce radicals by loss of a hydrogen
atom with that of anthralin, we determined the extent of the
compounds to scavenge radical intermediates by measuring
spectrophotometrically the decrease of DPPH concentration [25] at
520 nm. When DPPH reacts with an electron-donating test
compound (i.e., a hydrogen atom donor), it is reduced to the
corresponding hydrazine, whereas the primary species derived
from the test compound is a free radical.
Cl
Cl
Cl
Cl
R
R
5a–5x
7a–7c
Cpd
R
AAa
IC50
LDHb
DPPHd
(mM) (mU/mL) EC50 (mM)
1
3
0.7
122.8c
64.4
69.6
16.7
29.5
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
6.3
4,5-dichloro-10H-anthracen-9-one 2.1
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
4.3
44
25
30
36
>50
2.0
5.0
5.0
3.8
24
>50
>50
5.0
>50
17
22
36
>50
>50
15
19
18
0.7
>50
>50
20
e
4-CH3
e
e
e
e
4-CF3
2-Cl
4-Cl
2-OMe
3-OMe
4-OMe
3,4-(OMe)2
2,5-(OMe)2
3,5-(OMe)2
3,4,5-(OMe)3
2-NO2
3-NO2
4-NO2
2,4-(NO2)2
3-OBn
4-OBn
65.0
57.6
68.4
5j
5k
5l
60.6
e
e
e
4. Results and discussion
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
7a
7b
7c
69.9
e
4.1. Inhibition of keratinocyte hyperproliferation
e
e
e
e
e
e
e
The novel anthrone analogues of 3 were examined for inhibitory
action against hyperproliferation of HaCaT keratinocytes, as
demonstrated by reduction in cell number over time as compared
to control plates. The concentrations required to inhibit 50% of
keratinocyte hyperproliferation are shown in Table 1. A total of 27
compounds were tested, of which eight showed appreciable
3,4-(OBn)2
3-OBn-4-OMe
3-OH
4-OH
3,4-(OH)2
3-OH-4-OMe
H
76.6c
69.5
>50
>50
>50
>50
activity (IC50 value of 5
Starting with the basic 10-benzyl analogue 5a, which had an IC50
value of 4.3 M, various substituents were introduced in order to
mM or less).
e
e
e
Me
OMe
m
a
determine how they affected keratinocyte hyperproliferation. Our
data have shown that lipophilic and electron-withdrawing groups
substituted on the benzyl moiety generally caused a strong
decrease in the inhibition of keratinocyte growth compared to the
unsubstituted 5a. For instance, compounds 5bee, with methyl or
halogen substitution were considerably less potent than 5a. Also,
the nitro-substituted 5m, 5o and 5p were less active or inactive.
The only exception was 5n with a nitro group substituted at the
3-position of the benzyl moiety, which belonged to the active
analogues. Compounds 5uew explore the influence of hydroxy
groups, but there appeared to be no requirement for hydrogen
bond donor capability and the position at the phenyl ring, as these
compounds were less active than 5a and differed little in their
activity. By contrast, the corresponding methyl ethers 5gej showed
increased potency in the keratinocyte hyperproliferation assay in
comparison with the phenolic analogues. However, a 3,5-dime-
thoxy-substitution pattern in 5k, introduction of a third methoxy
group (5l) or larger benzyloxy groups as in 5qet all had detrimental
effects on activity. The substitution pattern that resulted in the
greatest improvement of potency was 3-hydroxy-4-methoxy,
providing a sixfold increase in inhibition of keratinocyte hyper-
proliferation over the unsubstituted analogue (5x versus 5a). With
Antiproliferative activity against keratinocytes. IC50, concentration of test
compound required for 50% inhibition of cell growth (HaCaT). Inhibition of cell
growth was significantly different with respect to that of the control, N ¼ 3, P < 0.01.
b
Activity of LDH (mU) release in HaCaT cells after treatment with 2
mM test
compound (N ¼ 3, SD < 10%).
c
Values are significantly different with respect to vehicle control (0.2% DMSO in
the culture medium, 70.6 mU/mL), P < 0.05. Brij 35 (polyoxyethyleneglycol dodecyl
ether)/ultrasound was the positive control (251 mU/mL).
d
DPPH radical interacting capability. EC50, effective concentration of test
compound (N ¼ 3, SD < 10%) required for 50% decrease in DPPH absorbance at
520 nm. NDGA (IC50 ¼ 4.2
m
M) and
a
-tocopherol (IC50 ¼ 8.2
mM) were used as
positive controls.
e
Not determined.
3. Biological assay methods
A rebalanced homeostatic control of keratinocyte growth and
differentiation is crucial for recovery from psoriatic to normal
epidermis. Therefore, compounds that ideally show low toxicity
and restore skin homeostasis by suppressing keratinocyte hyper-
proliferation are potentially useful in the treatment of psoriasis
[22].
In the present study, we used HaCaT keratinocytes [23] as
a model for the hyperproliferative epidermis in psoriasis. This
rapidly dividing, nontransformed human cell line is a useful tool in
an IC50 value in the submicromolar range (0.7
mM) and comparable
to that of standard anthralin, 5x was the most potent inhibitor of

U. Kratz et al. / European Journal of Medicinal Chemistry 45 (2010) 5278e5285
5281
keratinocyte hyperproliferation in this series. Even though the
5. Conclusions
mechanism of the antiproliferative action of 5x has not yet been
determined, a phenolic hydroxy group being flanked by one or two
methoxy groups was an important determinant for inhibitory
action against tubulin polymerization and usually related to strong
antiproliferative activity in anthrone derived inhibitors [26,27].
However, in these cases a double bond at C-10 was required and 10-
benzylated anthrones revealed a dramatic loss of antiproliferative
activity [18].
Finally, in the following compound series the methylene spacer
connecting the anthrone nucleus and the side chain aromatic ring
system was eliminated. Compound 7c showed weak anti-
proliferative activity while 7a and 7b were inactive. These findings
suggested that a higher flexibility with regard to the relative spatial
positions of the two ring systems might be important for activity.
Work aimed at further improving the benefit to risk ratio of the
anthrone class of antipsoriatic agents has led to 10-benzylated 4,5-
dichloro-10H-anthracen-9-ones devoid of radical interacting
properties. Structureeactivity relationship studies revealed that
lipophilic and electron-withdrawing substituents on the benzyl
moiety strongly decreased antiproliferative potency as compared to
the unsubstituted 10-benzyl derivative 5a whereas the methoxy
substituted compounds belonged to the most potent inhibitors of
keratinocyte hyperproliferation of this series. As a consequence and
in sharp contrast to anthralin, induction of membrane injury was
dramatically reduced as documented by the release of LDH activity
from cytoplasm of keratinocytes. The most promising member so
far, 3-hydroxy-4-methoxy-benzylated 5x, showed equally potent
inhibition of keratinocyte hyperproliferation as anthralin which
indicated therapeutic potential against hyperproliferative skin
diseases. As the substitution pattern of 5x is a typical pharmaco-
phore usually related to strong antiproliferative action and inhibi-
tion of tubulin polymerization within anthrone derived inhibitors,
this may reflect a possible target for its mode of action.
4.2. Integrity of plasma-membrane
The release of LDH is commonly used as an indicator of plasma-
membrane damage. In this assay, LDH release by the standard
anthralin significantly exceeded that of the vehicle control (0.2%
DMSO in the culture medium). On the other hand, the keratinocyte
growth inhibitory activity of the novel anthrone derivatives with
IC50 values in the low micromolar range was due to anti-
proliferative rather than non-specific cytotoxic effects, as LDH
6. Experimental section
release was unchanged as compared to controls at 2-
mM concen-
6.1. Chemistry
tration of the test compounds (Table 1). Catechol 5w was an
exception. Treatment of keratinocytes with this compound caused
a slight increase in LDH activity as compared to vehicle controls.
Most probably membrane damage produced by 5w is a result of the
radical generating properties of its catechol moiety. Aside from
parent anthralin, compound 5w was the only one that substantially
interacted with the free radical DPPH (see Section 4.3). This was
consistent with earlier observations that catechol moieties
enhanced formation of hydroxyl radicals [28].
Melting points were determined with a Kofler melting point
apparatus and are uncorrected. 1H NMR and 13C NMR spectra were
recorded with a Varian Gemini 200 (200 and 50.29 MHz, respec-
tively) spectrometer, using tetramethylsilane as an internal stan-
dard. Fourier-transform IR spectra (KBr) were recorded on a Bio-
Rad laboratories type FTS 135 spectrometer. Mass spectra were
obtained in the EI mode using a MAT GCQ Finnigan instrument.
Thin layer chromatography (TLC) was conducted on Merck 60 F254
precoated silica gel plates. Chromatography refers to column
chromatography, which was performed on Merck silica gel
(70e230 mesh) with CH2Cl2 as eluant, unless otherwise stated.
Yields have not been optimized. Elemental analyses were deter-
mined by the Microanalysis Laboratory at the University of
Münster, using a Vario EL III elemental analyzer. Elemental analyses
were within ꢁ0.4% of calculated values, except where stated
otherwise.
4.3. Interaction with the stable free radical DPPH
Table 1 shows that anthralin (1) itself proved to be very effective
in donating an electron to the stable free radical DPPH. In a similar
fashion, the unsubstituted parent structure of our novel analogues,
4,5-dichloro-9-anthrone (3), was also capable of effectively
reducing DPPH. This is in good agreement with our finding that this
agent is not stable in solution and in the keratinocyte culture
medium, and the primary radical generated by hydrogen abstrac-
tion from C-10 of the anthrone can dimerize in a radical recombi-
nation reaction to form the dimer 4. By contrast, with EC50 values
6.2. Stability of 4,5-dichloro-9-anthrone (3)
greater than 50
mM, 10-benzylated or phenylated analogues of
6.2.1. Materials
anthrone 3 were devoid of any appreciable radical interacting
activity in terms of their ineffectiveness to reduce the free radical
DPPH. Obviously, the 10-substituent stabilizes the molecules by
preventing hydrogen atom abstraction from the anthrones by a free
radical such as DPPH. These results were in excellent agreement
with our data obtained from the LDH assay in which these novel
compounds were devoid of membrane-damaging properties. The
only exception was 3,4-dihydroxy-substituted 5w, which was also
capable of interacting with the DPPH radical as effectively as the
Commercially available 1,8-dichloroanthracene-9,10-dione and
4,5-dichloro-10H-anthracen-9-one (3), prepared as described [20],
were purified by chromatography (SiO2/CH2Cl2).
6.2.1.1. 1,10,8,80-Tetrachloro-[9,90-bianthracene]-10,100(9H,90H)-dione
(dimer 4). To
a solution of 4,5-dichloro-10H-anthracen-9-one
(3, 0.25 g, 1.0 mmol) in boiling acetic acid (25 mL) was added
dropwise under light protection and N2 a solution of iron(III)
chloride (10%, 2 mL), and the solution was refluxed for 2 h. Then
water (25 mL) was added, and the mixture was extracted with
CH2Cl2 (2 ꢂ 50 mL). The combined organic phase was dried over
Na2SO4, concentrated in vacuo and purified by chromatography
(SiO2/CH2Cl2) to provide a yellow-green powder; 43% yield; mp
antioxidants NDGA or a-tocopherol, thus reflecting the reducing
capability of the catechol group.
The fact that the ability of the novel 10-substituted anthrones to
arrest keratinocyte hyperproliferation was not mediated by non-
specific redox properties or cytotoxic effects on the keratinocyte
membrane compares them favorably with anthralin as an alter-
native method for treating psoriasis.
297 ꢀC (Ref. [29] > 300 ꢀC); 1H NMR (CDCl3)
d 7.68 (4H, dd,
J1 ¼ 2.0 Hz, J2 ¼ 7.1 Hz), 7.39e7.27 (8H, m), 5.99 (2H, s); MS m/
z ¼ 524 (1, Mþ), 227 (100).

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U. Kratz et al. / European Journal of Medicinal Chemistry 45 (2010) 5278e5285
6.2.2. Sample preparation
6.2.4.4. 4,5-Dichloro-10-(2-chlorobenzyl)-10H-anthracen-9-one
Stock solutions of 3 (10 and 50
m
mol/L) were prepared in DMSO
(5d). The title compound was obtained from 3 (0.5 g, 2.0 mmol)
and 2-chlorobenzyl bromide [31] (1.7 g, 8.0 mmol) as described for
5a. Purification by chromatography (SiO2; CH2Cl2/hexane, 4/1) gave
(10 mL) as well as in Dulbecco’s modified Eagle’s medium (DMEM,
No. 041-11965A, Gibco) supplemented with 10% fetal calf serum,
penicillin (100 U/mL), streptomycin (100 mg/mL), and kept in
a humidified incubator containing 8% CO2 at 37 ꢀC. After 48 h of
incubation, each samples of 3 in DMEM were extracted with Et2O
(2 ꢂ 3 mL), the combined organic phase was dried over Na2SO4, and
then streamed with N2 to remove the solvent. The residue was
a yellow powder; 16% yield; mp 121e123 ꢀC; 1H NMR (CDCl3)
d 7.92
(2H, dd, J1 ¼ 1.1 Hz, J2 ¼ 7.74 Hz), 7.65 (2H, dd, J1 ¼ 1.4 Hz,
J2 ¼ 7.9 Hz), 7.39 (2H, t, J ¼ 7.8 Hz), 7.01 (2H, m), 6.73 (1H, m), 5.89
(1H, d, J ¼ 7.4 Hz), 5.50 (1H, t, J ¼ 4.7 Hz), 3.53 (2H, d, J ¼ 4.9 Hz);
FTIR 1671 cmꢃ1 (CO); MS m/z ¼ 386 (22, Mþ), 261 (100). Anal.
C21H13Cl3O (C, H).
dissolved in DMSO (50
mL). Finally, samples (50 mL) of stock solu-
tions in DMSO and those obtained from incubation in DMEM were
diluted with bidistilled water (100
m
L).
6.2.4.5. 4,5-Dichloro-10-(4-chlorobenzyl)-10H-anthracen-9-one
(5e). The title compound was obtained from 3 (0.5 g, 2.0 mmol) and
4-chlorobenzyl bromide [32] (1.7 g, 8.0 mmol) as described for 5a.
Purification by chromatography (SiO2; CH2Cl2/hexane, 4/1) gave
6.2.3. HPLC analysis
HPLC analysis was performed on a chromatographic system
(Merck/Hitachi) equipped with an L-6200A intelligent pump,
Rheodine injector, 655A variable wavelength UV monitor, and
D2500 chromato-integrator; HibarÒ RT 125 ꢂ 4-mm column
a yellow powder; 23% yield; mp 130e132 ꢀC; 1H NMR (CDCl3)
d 7.94
(2H, d, J ¼ 7.8 Hz), 7.71 (2H, d, J ¼ 7.8 Hz), 7.39 (2H, t, J ¼ 7.8 Hz), 6.82
(2H, d, J ¼ 8.2 Hz), 5.92 (2H, d, J ¼ 8.2 Hz), 5.38 (1H, t, J ¼ 4.4 Hz), 3.39
packed with LiChrospher RP-18 (5-
m
m particles; Merck 50983,
L; the isocratic
(2H, d, J ¼ 4.5 Hz); 13C NMR (CDCl3)
d 182.3 (C]O),140.0,137.8,134.8,
Darmstadt, Germany). The injection volume was 50
m
133.4, 133.3, 133.1, 133.0, 126.2 (8C, Ar), 134.0, 131.4, 128.3, 127.8,
125.8 (10CH, Ar), 39.9, 39.1 (CH, CH2); FTIR 1685 cmꢃ1 (CO); MS
m/z ¼ 386 (11, Mþ), 125 (100). Anal. C21H13Cl3O (C, H).
elution conditions were MeCN/water (75/25), flow rate 1.0 mL/min,
monitored at 254 nm. With the isocratic HPLC conditions
employed, the retention times of 1,8-dichloroanthracene-9,10-
dione, 4,5-dichloro-9-anthrone (3), and dimer 4 were 3.38, 5.16,
and 14.90 min, respectively.
6.2.4.6. 4,5-Dichloro-10-(2-methoxybenzyl)-10H-anthracen-9-one
(5f). The title compound was obtained from 3 (0.33 g, 1.25 mmol)
and 2-methoxybenzyl bromide [33] (1.0 g, 5.0 mmol) as described
for 5a. Purification by chromatography (SiO2; CH2Cl2) gave yellow
6.2.4. General procedure for the benzylation of 4,5-dichloro-9-
anthrone
needles; 21% yield; mp 134e135 ꢀC; 1H NMR (CDCl3)
d 7.87 (2H, d,
6.2.4.1. 10-Benzyl-4,5-dichloro-10H-anthracen-9-one (5a). To a solu-
tion of 4,5-dichloro-10H-anthracen-9-one [20] (3, 0.25 g, 1.0 mmol)
and benzyl bromide (0.51 g, 3.0 mmol) in CH2Cl2 (20 mL) was added
a solution of TEBAC (0.50 g, 2.20 mmol) in 30% NaOH (15 mL), and
the mixture was stirred at RT for 2 h. Then water (50 mL) was
added, and the mixture was extracted with CH2Cl2 (2 ꢂ 25 mL). The
combined organic phase was dried over Na2SO4, concentrated in
vacuo, and the residue was purified by chromatography (SiO2;
CH2Cl2) to provide yellow crystals; 48% yield; mp 133e134 ꢀC
J ¼ 7.8 Hz), 7.67 (2H, d, J ¼ 8.0 Hz), 7.34 (2H, t, J ¼ 8.0 Hz), 7.04 (1H,
t, J ¼ 6.6 Hz), 6.44 (2H, t, J ¼ 8.3 Hz), 5.78 (1H, d, J ¼ 7.4 Hz), 5.45
(1H, t, J ¼ 4.3 Hz), 3.46 (2H, d, J ¼ 4.4 Hz), 3.09 (3H, s); FTIR
1670 cmꢃ1 (CO); MS m/z ¼ 382 (2, Mþ), 121 (100). Anal.
C22H16Cl2O2 (C, H).
6.2.4.7. 4,5-Dichloro-10-(3-methoxybenzyl)-10H-anthracen-9-one
(5g). The title compound was obtained from 3 (0.5 g, 2.0 mmol)
and 3-methoxybenzyl bromide [33] (1.0 g, 5.0 mmol) as
described for 5a. Recrystallization from hexane/petroleum ether
(1/1) gave pale-yellow crystals; 29% yield; mp 139e141 ꢀC; 1H
(Ref. [30] 148 ꢀC); 1H NMR (CDCl3)
d
7.89 (2H, dd, J1 ¼ 1.1 Hz,
J2 ¼ 7.8 Hz), 7.70 (2H, dd, J1 ¼ 1.2 Hz, J2 ¼ 7.9 Hz), 7.37 (2H, t,
J ¼ 7.9 Hz), 7.03 (1H, t, J ¼ 7.3 Hz), 6.83 (2H, t, J ¼ 7.6 Hz), 5.96 (2H, d,
J ¼ 7.3 Hz), 5.41 (1H, t, J ¼ 4.4 Hz), 3.4 (2H, d, J ¼ 4.4 Hz); FTIR
1666 cmꢃ1 (CO); MS m/z ¼ 352 (18, Mþ), 91 (100). Anal. C21H14Cl2O
(C, H).
NMR (CDCl3)
d
7.92 (2H, dd, J1 ¼ 1.3 Hz, J2 ¼ 7.8 Hz), 7.70 (2H, dd,
J1 ¼1.3 Hz, J2 ¼ 8.0), 7.38 (2H, t, J ¼ 7.9 Hz), 6.76 (1H, t, J ¼ 7.8 Hz),
6.58 (1H, dd, J1 ¼ 1.5 Hz, J2 ¼ 8.2 Hz), 5.59 (1H, d, J ¼ 7.4 Hz), 5.48
(1H, t, J ¼ 2.0 Hz), 5.42 (1H, t, J ¼ 4.4 Hz), 3.42 (3H, s) 3.38 (2H, d,
J ¼ 4.4 Hz); FTIR 1685 cmꢃ1 (CO); MS m/z ¼ 382 (12, Mþ), 121
(100). Anal. C22H16Cl2O2 (C, H).
6.2.4.2. 4,5-Dichloro-10-(4-methylbenzyl)-10H-anthracen-9-one
(5b). The title compound was obtained from 3 (0.5 g, 2.0 mmol)
and 4-methylbenzyl bromide (1.42 g, 8.0 mmol) as described for 5a.
Recrystallization from MeOH gave pale-yellow crystals; 30% yield;
6.2.4.8. 4,5-Dichloro-10-(4-methoxybenzyl)-10H-anthracen-9-one
(5h). The title compound was obtained from 3 (0.5 g, 2.0 mmol)
and 4-methoxybenzyl bromide [32] (1.0 g, 5.0 mmol) as described
for 5a. Recrystallization from hexane/petroleum ether (1/1) gave
pale-yellow crystals; 29% yield; mp 142e143 ꢀC; 1H NMR (CDCl3)
mp 153 ꢀC; 1H NMR (CDCl3)
d
7.90 (2H, dd, J1 ¼ 1.2 Hz, J2 ¼ 7.8 Hz),
7.70 (2H, dd, J1 ¼1.2 Hz, J2 ¼ 7.9 Hz), 7.37 (2H, t, J ¼ 7.8 Hz), 6.64 (2H,
d, J ¼ 7.7 Hz), 5.84 (2H, d, J ¼ 7.9 Hz), 5.39 (1H, t, J ¼ 4.5 Hz), 3.37
(2H, d, J ¼ 4.4 Hz), 2.16 (3H, s); FTIR 1670 cmꢃ1 (CO); MS m/z ¼ 366
(2.46, Mþ), 105 (100). Anal. C22H16Cl2O (C, H).
d
7.92 (2H, dd, J1 ¼ 1.1 Hz, J2
¼
7.8 Hz), 7.70 (2H, dd,
J1 ¼ 1.3 Hz, J2 ¼ 7.9 Hz), 7.37 (2H, t, J ¼ 789 Hz), 6.37 (2H, dd,
J1 ¼ 2.0 Hz, J2 ¼ 6.7 Hz), 5.86 (2H, dd, J1 ¼ 1.9 Hz, J2 ¼ 6.7 Hz), 5.39
(1H, t, J ¼ 4.3 Hz), 3.65 (3H, s), 3.34 (2H, d, J ¼ 4.4 Hz); FTIR
1672 cmꢃ1 (CO); MS m/z ¼ 382 (1, Mþ), 121 (100). Anal. C22H16Cl2O2
(C, H).
6.2.4.3. 4,5-Dichloro-10-(4-trifluoromethylbenzyl)-10H-anthracen-
9-one (5c). The title compound was obtained from 3 (0.5 g,
2.0 mmol) and 4-trifluoromethylbenzyl bromide (0.95 g,
8.0 mmol) as described for 5a. Purification by chromatography
(SiO2; CH2Cl2/hexane, 4/1) gave pale-yellow crystals; 12%
6.2.4.9. 4,5-Dichloro-10-(3,4-dimethoxybenzyl)-10H-anthracen-9-
one (5i). The title compound was obtained from 3 (0.5 g,
yield; mp 131e132 ꢀC; 1H NMR (CDCl3)
d
7.94 (2H, dd,
J1 ¼ 1.0 Hz, J2 ¼ 7.8 Hz), 7.72 (2H, dd, J1 ¼ 1.2 Hz, J2 ¼ 7.9 Hz),
7.38 (2H, t, J ¼ 7.9 Hz), 7.11 (2H, d, J ¼ 8.0 Hz), 6.15 (2H, d,
J ¼ 8.1 Hz), 5.43 (1H, t, J ¼ 4.6 Hz), 3.45 (2H, d, J ¼ 4.6 Hz); FTIR
1670 cmꢃ1 (CO); MS m/z ¼ 420 (14, Mþ), 261 (100). Anal.
C22H13Cl2F3O (C, H).
2.0 mmol) and 3,4-dimethoxybenzyl bromide [34] (1.8 g,
8.0 mmol) as described for 5a. Purification by chromatography
(SiO2; CH2Cl2/hexane, 4/1) and recrystallization from hexane/
petroleum ether/MeOH (1/1/1) gave a yellow powder; 13% yield;
mp 153 ꢀC; 1H NMR (CDCl3)
d
7.93 (2H, dd, J1 ¼ 1.1 Hz,

U. Kratz et al. / European Journal of Medicinal Chemistry 45 (2010) 5278e5285
5283
J2 ¼ 7.8 Hz), 7.70 (2H, dd, J1 ¼ 1.2 Hz, J2 ¼ 7.9 Hz), 7.37 (2H, t,
J ¼ 7.7 Hz), 6.35 (1H, d, J ¼ 8.2 Hz), 5.54 (1H, dd, J1 ¼ 1.9 Hz,
J2 ¼ 8.1 Hz), 5.43e5.38 (2H, m, 1H), 3.72 (3H, s), 3.36e3.33 (5H,
m); FTIR 1685 cmꢃ1 (CO); MS m/z ¼ 412 (2, Mþ), 151 (100). Anal.
C23H18Cl2O3 (C, H).
6.2.4.15. 4,5-Dichloro-10-(4-nitrobenzyl)-10H-anthracen-9-one
(5o). The title compound was obtained from 3 (0.5 g, 2.0 mmol)
and 4-nitrobenzyl bromide (0.7 g, 4.0 mmol) as described for 5a.
Purification by chromatography (SiO2; CH2Cl2) gave an orange
powder; 46% yield; mp 199e200 ꢀC; 1H NMR (CDCl3)
d 7.90 (2H, dd,
J1 ¼ 1.2 Hz, J2 ¼ 7.7 Hz), 7.66 (2H, dd, J1 ¼ 1.4 Hz, J2 ¼ 8.0 Hz), 7.56
(1H, dd, J1 ¼1.3 Hz, J2 ¼ 8.1 Hz), 7.41 (2H, t, J ¼ 7.9 Hz), 7.23 (1H, dd,
J1 ¼ 1.4 Hz, J2 ¼ 7.7 Hz), 7.05 (1H, dd, J1 ¼ 1.4 Hz, J2 ¼ 7.5 Hz), 6.07
(1H, dd, J1 ¼ 1.3 Hz, J2 ¼ 7.7 Hz), 5.48 (1H, t, J ¼ 5.0 Hz), 3.87
6.2.4.10. 4,5-Dichloro-10-(2,5-dimethoxybenzyl)-10H-anthracen-9-
one (5j). The title compound was obtained from 3 (0.22 g,
0.8 mmol) and 2,5-dimethoxybenzyl bromide [35] (0.7 g,
3.0 mmol) as described for 5a. Recrystallization from MeOH
gave pale-yellow crystals; 31% yield; mp 167e168 ꢀC; 1H NMR
(2H, d, J ¼ 4.9 Hz); 13C NMR (CDCl3)
d 182.2 (C]O), 146.9, 142.8,
139.5, 134.6, 133.1 (8C, Ar), 134.2, 130.9, 128.7, 126.0, 122.7 (10CH,
Ar), 40.4, 38.8 (CH, CH2); FTIR 1664 cmꢃ1 (CO); MS m/z ¼ 397 (3,
Mþ), 261 (100). Anal. C21H13Cl2NO3 (C, H, N).
(CDCl3)
d
7.89 (2H, d, J ¼ 7.7 Hz), 7.62 (2H, d, J ¼ 7.8 Hz), 7.34
(2H, t, J ¼ 7.9 Hz), 6.58 (1H, dd, J1 ¼ 3.0 Hz, J2 ¼ 8.9 Hz), 6.33
(1H, d, J ¼ 8.9 Hz), 5.44 (1H, t, J ¼ 4.4 Hz), 5.36 (1H, d,
J ¼ 2.0 Hz), 3.42 (2H, d, J ¼ 4.6 Hz), 3.39 (3H, s), 3.04 (3H, s);
FTIR 1668 cmꢃ1 (CO); MS m/z ¼ 412 (6, Mþ), 151 (100). Anal.
C23H18Cl2O3 (C, H).
6.2.4.16. 4,5-Dichloro-10-(2,4-dinitrobenzyl)-10H-anthracen-9-one
(5p). The title compound was obtained from 3 (0.25 g, 1.0 mmol)
and 2,4-dinitrobenzyl bromide (0.9 g, 4.0 mmol) as described for
5a. Purification by chromatography (SiO2; CH2Cl2hexane, 4/1) gave
6.2.4.11. 4,5-Dichloro-10-(3,5-dimethoxybenzyl)-10H-anthracen-9-
one (5k). The title compound was obtained from 3 (0.5 g,
2.0 mmol) and 3,5-dimethoxybenzyl bromide [36] (1.8 g, 8.0 mmol)
as described for 5a. Purification by chromatography (SiO2; CH2Cl2)
gave a pale-yellow powder; 13% yield; mp 165 ꢀC; 1H NMR (CDCl3)
a yellow powder; 31% yield; mp 179e180 ꢀC; 1H NMR (CDCl3)
d 8.44
(1H, d, J ¼ 2.3 Hz), 7.96 (2H, dd, J1 ¼1.0 Hz, J2 ¼ 7.8 Hz), 7.89 (1H, d,
J ¼ 2.5 Hz), 7.50 (2H, dd, J1 ¼ 1.3 Hz, J2 ¼ 8.0 Hz), 7.46 (2H, t,
J ¼ 7.8 Hz), 6.38 (1H, d, J ¼ 8.5 Hz), 5.10 (1H, t, J ¼ 5.3 Hz), 3.85
(2H, d, J ¼ 5.3 Hz); FTIR 1670 cmꢃ1 (CO); MS m/z ¼ 442 (1, Mþ), 261
(100). Anal. C21H12Cl2N2O5 (C, H, N).
d
7.95 (2H, dd, J1 ¼ 1.2 Hz, J2 ¼ 7.7 Hz), 7.71 (2H, dd, J1 ¼ 1.3 Hz,
J2 ¼ 7.9 Hz), 7.38 (2H, t, J ¼ 7.9 Hz), 6.13 (1H, t, J ¼ 2.3 Hz), 5.40 (1H, t,
J ¼ 4.4 Hz), 5.14 (2H, d, J ¼ 2.4 Hz), 3.41 (6H), 3.34 (2H, d, J ¼ 4.5 Hz);
FTIR 1676 cmꢃ1 (CO); MS m/z ¼ 412 (16, Mþ), 151 (100). Anal.
C23H18Cl2O3 (C, H).
6.2.4.17. 10-(3-Benzyloxybenzyl)-4,5-dichloro-10H-anthracen-9-one
(5q). The title compound was obtained from 3 (0.5 g, 2.0 mmol)
and 3-benzyloxybenzyl bromide [39] (1.1 g, 4.0 mmol) as
described for 5a. Purification by chromatography (SiO2; CH2Cl2)
gave a pale-yellow powder; 19% yield; mp 188e189 ꢀC; 1H NMR
6.2.4.12. 4,5-Dichloro-10-(3,4,5-trimethoxybenzyl)-10H-anthracen-
9-one (5l). The title compound was obtained from 3 (0.5 g,
2.0 mmol) and 3,4,5-trimethoxybenzyl bromide [37] (2.1 g,
8.0 mmol) as described for 5a. Recrystallization from MeOH/
petroleum ether (1/1) gave a yellow powder; 58% yield; mp
(CDCl3)
d
7.94 (2H, d, J ¼ 7.2 Hz), 7.70 (2H, dd, J1 ¼ 1.3 Hz,
J2 ¼ 7.9 Hz), 7.42e7.26 (7H, m), 6.80e6.62 (2H, m), 5.61 (2H, m),
5.42 (1H, t, J ¼ 4.4 Hz), 4.65 (2H, s), 3.38 (2H, d, J ¼ 4.6 Hz); FTIR
1669 cmꢃ1 (CO); MS m/z ¼ 458 (13, Mþ), 197 (100). Anal.
C28H20Cl2O2 (C, H).
156e157 ꢀC; 1H NMR (CDCl3)
d
7.93 (2H, dd, J1 ¼1.4 Hz, J2 ¼ 7.8 Hz),
7.71 (2H, dd, J1 ¼1.4 Hz, J2 ¼ 7.9 Hz), 7.38 (2H, t, J ¼ 7.9 Hz), 5.42 (1H,
t, J ¼ 4.4 Hz), 5.15 (2H, s), 3.72 (3H, s), 3.35e3.31 (8H, m); 13C NMR
6.2.4.18. 10-(4-Benzyloxybenzyl)-4,5-dichloro-10H-anthracen-9-one
(5r). The title compound was obtained from 3 (1.0 g, 4.0 mmol) and
4-benzyloxybenzyl bromide [39] (2.2 g, 8.0 mmol) as described for
5a. Purification by chromatography (SiO2; CH2Cl2) gave a yellow
(CDCl3) d 181.8 (C]O),152.3,140.3,137.6, 135.2,133.1,129.9 (6C, Ar),
133.7, 128.0, 125.5, 107.7 (8CH, Ar), 61.2 (OCH3), 55.8 (2 OCH3), 40.2,
39.3 (CH, CH2); FTIR 1676 cmꢃ1 (CO); MS m/z ¼ 442 (2, Mþ), 181
(100). Anal. C24H20Cl2O4 (C, H).
powder; 47% yield; mp 192e193 ꢀC; 1H NMR (CDCl3)
d 7.92 (2H, d,
J ¼ 7.6 Hz), 7.69 (2H, d, J ¼ 7.8 Hz), 7.40e7.30 (7H, m), 6.44 (2H, d,
J ¼ 8.5 Hz), 5.87 (2H, d, J ¼ 8.5 Hz), 5.39 (1H, t, J ¼ 4.3 Hz), 4.90
(2H, s), 3.34 (2H, d, J ¼ 4.4 Hz); FTIR 1669 cmꢃ1 (CO); MS m/z ¼ 458
(1, Mþ), 91 (100). Anal. C28H20Cl2O2 (C, H).
6.2.4.13. 4,5-Dichloro-10-(2-nitrobenzyl)-10H-anthracen-9-one
(5m). The title compound was obtained from 3 (0.25 g, 1.0 mmol)
and 2-nitrobenzyl bromide (0.7 g, 4.0 mmol) as described for 5a.
Purification by chromatography (SiO2; CH2Cl2/hexane, 1/3) gave
a yellow powder; 36% yield; mp 175e176 ꢀC; 1H NMR (CDCl3)
d
7.90
6.2.4.19. 10-[3,4-Bis(benzyloxy)benzyl]-4,5-dichloro-10H-anthracen-
9-one (5s). Thetitlecompoundwas obtainedfrom 3 (0.5g, 2.0 mmol)
and 3,4-bis(benzyloxy)benzyl bromide [39] (1.5 g, 3.9 mmol) as
described for 5a. Purification by chromatography (SiO2; CH2Cl2/
hexane, 4/1) gave a pale-yellow powder; 20% yield; mp 114e115 ꢀC;
(2H, dd, J1 ¼1.2 Hz, J2 ¼ 7.7 Hz), 7.66 (2H, dd, J1 ¼1.4 Hz, J2 ¼ 8.0 Hz),
7.56 (1H, dd, J1 ¼1.3 Hz, J2 ¼ 8.1 Hz), 7.41 (2H, t, J ¼ 7.9 Hz), 7.23 (1H,
dd, J1 ¼1.4 Hz, J2 ¼ 7.7 Hz), 7.05 (1H, dd, J1 ¼1.4 Hz, J2 ¼ 7.5 Hz), 6.07
(1H, dd, J1 ¼ 1.3 Hz, J2 ¼ 7.7 Hz), 5.48 (1H, t, J ¼ 5.0 Hz), 3.87 (2H, d,
J ¼ 4.9 Hz); FTIR 1668 cmꢃ1 (CO); MS m/z ¼ 397 (7, Mþ), 261 (100).
Anal. C21H13Cl2NO3 (C, H, N).
1H NMR (CDCl3)
d
7.93 (2H, d, J ¼ 7.8 Hz), 7.68 (2H, d, J ¼ 8.0 Hz),
7.39e7.25 (12H, m), 6.41 (1H, d, J ¼ 8.1 Hz), 5.56 (2H, m), 5.37 (1H, t,
J ¼ 4.4 Hz), 4.99 (2H, s), 4.60 (2H, s), 3.30 (2H, d, J ¼ 4.4 Hz); FTIR
1670 cmꢃ1 (CO);MSm/z¼ 564(1, Mþ),181(100).Anal. C35H26Cl2O3 (C,
H).
6.2.4.14. 4,5-Dichloro-10-(3-nitrobenzyl)-10H-anthracen-9-one
(5n). The title compound was obtained from 3 (0.25 g, 1.0 mmol)
and 3-nitrobenzyl bromide [38] (0.7 g, 4.0 mmol) as described for
5a. Purification by chromatography (SiO2; CH2Cl2/hexane, 4/1) gave
a pale-yellow powder; 58% yield; mp 170e171 ꢀC; 1H NMR (CDCl3)
6.2.4.20. 10-(3-Benzyloxy-4-methoxybenzyl)-4,5-dichloro-10H-
anthracen-9-one (5t). The title compound was obtained from 3
(0.5 g, 2.0 mmol) and 3-benzyloxy-4-methoxybenzyl bromide [40]
(1.2 g, 3.9 mmol) as described for 5a. Purification by chromatog-
raphy (SiO2; CH2Cl2/hexane, 4/1) gave a pale-brownish powder;
d
7.92 (1H, dd, J1 ¼ 1.0 Hz, J2
¼
8.3 Hz), 7.90 (2H, dd,
J1 ¼ 1.3 Hz, J2 ¼ 7.8 Hz), 7.76 (2H, dd, J1 ¼ 1.4 Hz, J2 ¼ 7.9 Hz), 7.42
(2H, t, J ¼ 8.0 Hz), 7.03 (1H, t, J ¼ 7.9 Hz), 6.83 (1H, t, J ¼ 1.9 Hz), 6.32
(1H, d, J ¼ 7.6 Hz), 5.47 (1H, t, J ¼ 4.5 Hz), 3.50 (2H, d, J ¼ 4.7); FTIR
1669 cmꢃ1 (CO); MS m/z ¼ 397 (12, Mþ), 261 (100). Anal.
C21H13Cl2NO3 (C, H, N).
59% yield; mp 139e140 ꢀC; 1H NMR (CDCl3)
d 7.88 (2H, dd,
J1 ¼ 1.1 Hz, J2 ¼ 7.8 Hz), 7.62 (2H, d, J1 ¼ 1.2 Hz, J2 ¼ 7.9 Hz), 7.25
(7H, m), 6.33 (1H, d, J ¼ 8.8 Hz), 5.49 (2H, m), 5.29 (1H, t,

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U. Kratz et al. / European Journal of Medicinal Chemistry 45 (2010) 5278e5285
J ¼ 4.3 Hz), 4.50 (2H, s), 3.65 (3H, s), 3.22 (2H, d, J ¼ 4.3 Hz); FTIR
1670 cmꢃ1 (CO); MS m/z ¼ 488 (2, Mþ), 227 (100). Anal.
C29H22Cl2O3 (C, H).
mp 238e239 ꢀC (Ref. [21] 237 ꢀC); 1H NMR (CDCl3)
d 8.25 (2H, dd,
J1 ¼ 1.4 H, J2 ¼ 7.7 Hz), 7.58 (2H, dd, J1 ¼ 1.4 Hz, J2 ¼ 8.00 Hz), 7.39
(2H, t, J ¼ 7.9 Hz), 7.29e7.09 (5H, m), 6.11 (1H, s); FTIR 1673 cmꢃ1
(CO); MS m/z ¼ 338 (100, Mþ). Anal. C20H12Cl2O (C, H).
6.2.5. General procedure for the hydrogenolytic cleavage of benzyl
ethers
6.2.6.2. 4,5-Dichloro-10-(4-methylphenyl)-10H-anthracen-9-one
(7b). The title compound was obtained from 6 (0.1 g, 0.3 mmol) in
toluene (0.87 g, 9.4 mmol) as described for 7a. Purification by
chromatography (SiO2; CH2Cl2) gave a white powder; 78% yield;
6.2.5.1. 4,5-Dichloro-10-(3-hydroxybenzyl)-10H-anthracen-9-one
(5u). A solution of 5q (0.1 g, 0.6 mmol) in absolute THF (50 mL) was
hydrogenated over 10% PdeC (0.15 g) at RT and atmospheric pres-
sure. After the requisite amount of H2 had been taken up (12 h,
TLC-control), the catalyst was removed by filtration and the solution
was evaporated. The product was recrystallized from hexane/
petroleum ether (1/1) and purified by chromatography (SiO2; MeOH)
to provide a white powder; 9% yield; mp 211e212 ꢀC; 1H NMR
mp 241e242 ꢀC; 1H NMR (CDCl3)
d
8.23 (2H, dd,
J1 ¼ 1.5 Hz, J2 ¼ 7.8 Hz), 7.57 (2H, dd, J1 ¼ 1.4 Hz, J2 ¼ 7.9 Hz), 7.35
(2H, t, J ¼ 7.8 Hz), 7.15 (2H, d, J ¼ 8.2 Hz), 6.80 (2H, d, J ¼ 8.0 Hz), 6.08
(1H, s), 2.22 (3H, s); FTIR 1676 cmꢃ1 (CO); MS m/z ¼ 352 (100, Mþ).
Anal. C21H14Cl2O (C, H).
(CDCl3)
d
7.93 (2H, dd, J1 ¼1.1 Hz, J2 ¼ 7.8 Hz), 7.70 (2H, dd, J1 ¼1.2 Hz,
J2 ¼ 8.0 Hz), 7.38 (2H, t, J ¼ 7.9 Hz), 6.70 (1H, t, J ¼ 7.8 Hz), 6.49 (1H, d,
6.2.6.3. 4,5-Dichloro-10-(4-methoxyphenyl)-10H-anthracen-9-one
(7c). The title compound was obtained from 6 (0.5 g, 1.5 mmol) in
anisole (0.5 g, 4.6 mmol) and AlCl3 (0.5 g, 3.8 mmol) as described for
7a, but the suspension was refluxed for 2 h. The crude product was
extracted with CH2Cl2 (3 ꢂ 50 mL), the combined organic phase
dried over Na2SO4, and evaporated. Purification by chromatography
(SiO2; CH2Cl2) gave a white-yellow powder; 73% yield; mp 240 ꢀC;
J ¼ 7.7 Hz), 5.57e5.39 (3H, m), 4.36 (1H, s), 3.36 (2H, d, J ¼ 4.4 Hz);
13C NMR (CDCl3)
d 154.9, 140.3, 136.3, 135.0, 133.0, 130.3, 127.6, 126.8
(8C, Ar), 133.9, 128.7, 128.3, 125.5, 122.7, 117.4, 113.8 (10CH, Ar), 40.1,
39.1 (CH, CH2); FTIR 1657 cmꢃ1 (CO); MS m/z ¼ 368 (1, Mþ), 262
(100). Anal. C21H14Cl2O2 (C, H).
6.2.5.2. 4,5-Dichloro-10-(4-hydroxybenzyl)-10H-anthracen-9-one
(5v). The title compound was obtained from 5r (0.2 g, 1.27 mmol)
as described for 5u. The product was recrystallized from hexane/
petroleum ether (1/1) and purified by chromatography (SiO2;
1H NMR (CDCl3)
d
8.24 (2H, dd, J1 ¼1.1 Hz, J2 ¼ 7.9 Hz), 7.57 (2H, dd,
J1 ¼ 1.4 Hz, J2 ¼ 7.9 Hz), 7.38 (2H, t, J ¼ 7.8 Hz), 7.17 (2H, dd,
J1 ¼ 2.2 Hz, J2 ¼ 6.7 Hz), 6.71 (2H, dd, J1 ¼ 2.08 Hz, J2 ¼ 6.71 Hz), 6.05
(1H, s), 3.69 (3H, s); FTIR 1672 cmꢃ1 (CO); MS m/z ¼ 368 (100, Mþ).
Anal. C21H14Cl2O2 (C, H).
MeOH) to provide
a
pale-yellow powder; 17% yield; mp
7.90 (2H, d, J ¼ 7.7 Hz), 7.72 (2H, d,
210e211 ꢀC; 1H NMR (CDCl3)
d
J ¼ 7.8 Hz), 7.37 (2H, t, J ¼ 7.8 Hz), 6.35 (2H, d, J ¼ 8.2 Hz), 5.80
(2H, d, J ¼ 8.2 Hz), 5.66 (1H, s), 5.41 (1H, t, J ¼ 4.1 Hz), 3.35 (2H, d,
J ¼ 4.3 Hz); FTIR 1650 cmꢃ1 (CO); MS m/z ¼ 368 (1, Mþ), 262 (100).
Anal. C21H14Cl2O2 (C, H).
6.3. Biological assay methods
6.3.1. Keratinocyte culture and determination of cell growth
HaCaT cells [23] were cultivated and the cell proliferation assay
was performed as described [28]. After 48 h of incubation, cell
growth was determined by enumerating the dispersed cells by
phase-contrast microscopy. Inhibition was calculated by the
comparison of the mean values of the test compound (N ¼ 3) with
the control (N ¼ 6e8) activity: (1 ꢃ test compound/control) ꢂ 100.
Inhibition was statistically significant compared to that of the
control (Student’s t-test; P < 0.05). IC50 values were obtained by
nonlinear regression.
6.2.5.3. 4,5-Dichloro-10-(3,4-dihydroxybenzyl)-10H-anthracen-9-
one (5w). The title compound was obtained from 5s (0.15 g,
0.27 mmol) as described for 5u. Recrystallization from hexane/
petroleum ether (1/1) gave a pale-yellow powder; 30% yield; mp
161e162 ꢀC; 1H NMR (CDCl3)
d
7.87 (2H, d, J ¼ 7.7 Hz), 7.71 (2H, d,
J ¼ 7.7 Hz), 7.36 (2H, t, J1 ¼ J2 ¼ 7.8 Hz), 6.35 (1H, d, J ¼ 7.9 Hz), 6.15
(1H, s), 5.48e5.35 (4H, m), 3.30 (2H, d, J ¼ 4.2 Hz); 13C NMR (CDCl3)
d
183.8 (C]O), 143.3, 142.8, 140.6, 135.0, 133.0, 128.5, 126.9, 125.8,
112.4 (9C, Ar), 134.1, 128.2, 125.4, 122.6, 117.6, 114.6 (9CH, Ar), 39.9,
39.4 (CH, CH2); FTIR 1656 cmꢃ1 (CO); MS m/z ¼ 384 (1, Mþ), 262
(100). Anal. C21H14Cl2O3 (C, H).
6.3.2. Lactate dehydrogenase release
The assay was performed as described [24]. HaCaT cells were
incubated with the test compounds (2 m
M) for 4 h at 37 ꢀC. Extra-
cellular LDH activity was measured using the UV method with
pyruvate and NADH and is expressed in mU/ml. Appropriate
controls with the vehicle were performed (P < 0.01; N ¼ 3,
SD < 10%). Brij 35 (polyoxyethyleneglycol dodecyl ether)/ultra-
sound was the positive control.
6.2.5.4. 4,5-Dichloro-10-(3-hydroxy-4-methoxybenzyl)-10H-anthra-
cen-9-one (5x). The title compound was obtained from 5t (0.2 g,
0.41 mmol) as described for 5u. Recrystallization from hexane/
petroleum ether (1/1) gave a yellow powder; 57% yield; mp
137e138 ꢀC; 1H NMR (CDCl3)
d
7.95 (2H, dd, J1 ¼1.2 Hz, J2 ¼ 7.8 Hz),
7.71 (2H, dd, J1 ¼1.3 Hz, J2 ¼ 7.9 Hz), 7.37 (2H, t, J ¼ 7.8 Hz), 6.30 (1H,
d, J ¼ 8.1 Hz), 5.55 (1H, d, J ¼ 2.0 Hz), 5.41 (1H, dd, J1 ¼ 2.2 Hz,
J2 ¼ 8.2 Hz), 5.37 (1H, t, J ¼ 4.3 Hz), 5.30 (1H, s), 3.72 (3H, s), 3.31
(2H, d, J ¼ 4.5 Hz); FTIR 1671 cmꢃ1 (CO); MS m/z ¼ 398 (2, Mþ), 137
(100). Anal. C22H16Cl2O3 (C, H).
6.3.3. Determination of DPPH radical interacting capability
The assay was performed as described [41]. The capability to
interact with DPPH radical (%) was calculated by the comparison of
the mean values of the test compound (N ¼ 3) with those of the
control (N ¼ 8): (1 ꢃ absorbance of test compound/absorbance of
control) ꢂ 100. Results were expressed as EC50, the effective
concentration of the test compound required for 50% decrease in
DPPH absorbance at 520 nm. Values were obtained by nonlinear
regression.
6.2.6. General procedure for the preparation of 10-phenyl-
substituted 4,5-dichloro-9-anthrones
6.2.6.1. 4,5-Dichloro-10-phenyl-10H-anthracen-9-one (7a). To a sus-
pension of 10-bromo-4,5-dichloro-10H-anthracen-9-one [21] (6,
0.1 g, 0.3 mmol) in benzene (0.88 g, 11.3 mmol) was added AlCl3
(0.1 g, 0.75 mmol). The mixture was refluxed for 30 min, then
cooled, treated with HCl (1 mol/L, 10 mL) and petroleum ether
(10 mL), and the product was filtered by suction. Purification by
chromatography (SiO2; CH2Cl2) gave a white powder; 40% yield;
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