Antipsoriatic anthrones with modulated redox properties. 5. Potent inhibition of human keratinocyte growth, induction of keratinocyte differentiation, and reduced membrane damage by novel 10-arylacetyl-1,8-dihydroxy-9(10H)-anthracenones

Article abstract of DOI:10.1021/jm001073w

The synthesis and structure - Activity relationships (SARs) of a series of novel 10-arylacetyl-1,8-dihydroxy-9(10H)-anthracenones are described. Acylation of anthralin with either the appropriate arylacetyl chlorides or arylacetic acids in the presence of

Full text of DOI:10.1021/jm001073w

814
J . Med. Chem. 2001, 44, 814-821
An tip sor ia tic An th r on es w ith Mod u la ted Red ox P r op er ties. 5. P oten t In h ibition
of Hu m a n Ker a tin ocyte Gr ow th , In d u ction of Ker a tin ocyte Differ en tia tion , a n d
Red u ced Mem br a n e Da m a ge by Novel
10-Ar yla cetyl-1,8-d ih yd r oxy-9(10H)-a n th r a cen on es
Klaus Mu¨ller,*,‡ Hans Reindl,§ and Klaus Breu§
Institut fu¨r Pharmazeutische Chemie, Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Hittorfstrasse 58-62, D-48149 Mu¨nster,
Germany, and Institut fu¨r Pharmazie, Pharmazeutische Chemie I, Universita¨t Regensburg, D-93040 Regensburg, Germany
Received September 8, 2000
The synthesis and structure-activity relationships (SARs) of a series of novel 10-arylacetyl-
1,8-dihydroxy-9(10H)-anthracenones are described. Acylation of anthralin with either the
appropriate arylacetyl chlorides or arylacetic acids in the presence of pyridine or via the coupling
agent dicyclohexylcarbodiimide (DCC), respectively, furnished this structural class of antip-
soriatic agents. Potential antipsoriatic activity was evaluated in complementary assays
specifically addressed to three important aspects of psoriasis. First, several compounds were
identified which are equally potent as inhibitors of human keratinocyte growth as the
antipsoriatic agent anthralin. Furthermore, improved ratio of antiproliferative activity to
cytotoxicity is demonstrated by the reduced potential of the novel analogues to induce membrane
damage, which is a benefit of their reduced ability to generate oxygen radicals as documented
by deoxyribose degradation. Second, analogue 3o bearing a hydroxamate functional group was
also a highly potent inhibitor of LTB4 biosynthesis in addition to its excellent antiproliferative
activity. SARs of these inhibitors of both keratinocyte growth and LTB4 biosynthesis with respect
to the nature of the para-substitution in the 10-phenylacetyl side chain are discussed. Third,
the compounds were also evaluated for their ability to induce the formation of cornified envelope
protein in keratinocytes. Cross-linking of cellular protein as a marker of terminal differentiation
of keratinocytes was observed for many 10-arylacetyl analogues at concentrations required to
arrest cell growth. This newly uncovered activity of the novel anthracenones suggests
antipsoriatic potential with respect to disturbance of keratinocyte differentiation, in addition
to hyperproliferative and inflammatory aspects of psoriasis.
In tr od u ction
antipsoriatic efficacy is retained while proinflammatory
effects are minimized.8
Psoriasis is a common skin disease with a prevalence
of 1-2% of the population in northern Europe and North
America.1 Dominant and interdependent features of the
disease are epidermal hyperproliferation, disturbed
keratinocyte differentiation, and inflammation of the
dermis and epidermis.2 Current treatments for psoriasis
may be topical or systemic.1,3 Indications for systemic
treatment are failure to respond to topical treatment
and severe or life-threatening forms of psoriasis.4 More
recently, vitamin D analogues and vitamin A analogues
have been added to the traditional topical therapy, but
these agents serve only to abate the disease.5 Yet
anthracenones such as anthralin (1, dithranol) clear the
rash totally.6 Indeed, of all antipsoriatic agents, anthra-
lin has proven to be the most remarkably consistent and
time-honored drug for treating psoriasis.7 Because of the
undesirable side effects such as severe inflammation of
nonaffected skin associated with this topical agent,
sometimes successful courses of treatment must even
be halted. Perhaps the best solution of this problem
would be development of structural analogues in which
The methylene moiety at C-10 of anthralin has been
recognized as a key site of the formation of oxygen
radicals,9 which play a fundamental role in the induc-
tion of skin inflammation. Moreover, in addition to
directly damaging cellular components of the skin,
oxygen radicals are responsible for the induction of
inflammatory cytokines which, in turn, contributes to
the dermal toxicity of anthralin.10 Therefore, our strat-
egy to overcome this problem was to modulate the
oxygen-radical generating intensity by modifying the
critical 10-position of the pharmacophore. Since our
fundamental work with anthracenone-based antipsori-
atics established that the 1,8-dihydroxy substitution
pattern of anthralin is required for potent antiprolif-
erative activity,11 we decided to retain this feature in
our compounds. In our earlier report, we have estab-
lished 10-substituted derivatives as antipsoriatic an-
thracenones with modulated redox properties, and we
demonstrated their inhibitory activity against 5-lipoxy-
genase.12
In our continuing investigation of structure-activity
relationships (SARs) of the anthracenone class of anti-
psoriatic agents, we further studied the biological
properties of 10-substituted congeners of anthralin. In
this paper, we report the synthesis of novel 10-arylacetyl
* To whom correspondence should be addressed. Tel: +49 251-
8333324. Fax: +49 251-8332144. E-mail: kmuller@uni-muenster.de.
‡ Universita¨t Mu¨nster.
§ Universita¨t Regensburg.
10.1021/jm001073w CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/06/2001

Antipsoriatic Anthrones with Modulated Redox Properties
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 815
Sch em e 1a
Sch em e 2a
a
Reagents: (a) Na, EtOH, H2O, rt, 12 h; (b) K2CO3, 3-MeOBnCl,
KI, acetone, ∆, 12 h; (c) 6 N NaOH, ∆, 4 h.
a
Reagents: (a) method A, X ) Cl: pyridine, THF, N2; method
B, X ) OH: DCC, pyridine, THF, N2; (b) NH(Me)OH‚HCl, EDC,
DMF, N2; (c) THF, trifluoroacetic acid. R1 and R2 are defined in
Table 1.
3-methoxybenzyl chloride followed by saponification of
the resulting 4. Compounds 3k k , 3m m , 3n n , and 3tt
were prepared from the antiinflammatory arylacetic
acids diclofenac, ibuprofen, ketoprofen, and indometha-
cin, respectively.
analogues and the assessment of their potential value
as antipsoriatic agents in complementary assays specif-
ically addressed to three important aspects of psoriasis.
Thus, in addition to their leukotriene B4 (LTB4) inhibi-
tory action as a measure of their antiinflammatory
properties, we also evaluated their antiproliferative
activity against the growth of HaCaT keratinocytes and
we describe their potential to induce differentiation of
human keratinocytes. Finally, we examined their cyto-
toxicity in terms of membrane-damaging effects.
Biologica l Eva lu a tion a n d Discu ssion
The structures of the 10-arylacetyl substituted an-
thracenones are listed in Table 1, together with relevant
biological properties. The biological assay procedures
used were exactly as those described in our previous
studies.12,18,19 Thus, modulated redox properties in
terms of hydroxyl-radical generation were studied using
the deoxyribose assay.12 The release of malondialdehyde
(MDA) is indicative of hydroxyl-radical generation.
Anthralin (1) and compound 3a in Table 1 are the
parent anthracenone and the unsubstituted 10-phenyl-
acetyl analogue (R ) H), respectively. Comparison
documents the positive effect for the phenylacetyl sub-
stituent, which dramatically decreases hydroxyl-radical
formation.12 Table 1 shows that, with the exception of
a few compounds such as 3p and 3q, generation of
hydroxyl radicals is markedly reduced or not signifi-
cantly different with respect to controls.
In h ib it ion of Ker a t in ocyt e Gr ow t h a n d LTB4
Biosyn t h esis. The immortalized keratinocyte line
HaCaT20 was used to mimic the hyperproliferative
epidermis found in psoriasis, as antiproliferative action
in cell cultures may be critical in the management of
the proliferative component of psoriasis. Proliferation
of the keratinocytes was determined directly by counting
the dispersed cells under a phase-contrast microscope
after 48 h of treatment. Inhibition of LTB4 biosynthesis
by the novel compounds was determined by measuring
the production of LTB4 in bovine polymorphonuclear
leukocytes.
Ch em istr y
Some of the required para-substituted 10-phenyl-
acetyl analogues were available from previous work, and
these are referenced in Table 1. The novel 10-phenyl-
acetyl analogues were prepared by reaction of 1 with
appropriate phenylacetyl chlorides in the presence of
pyridine (Scheme 1, method A, X ) Cl), where acylation
takes place at the C-10 position via the carbanion.13 The
required phenylacetyl chlorides were prepared from the
corresponding phenylacetic acids 2 according to litera-
ture procedures.14,15 In an alternative method (method
B, X ) OH), phenylacetic acids 2 were directly intro-
duced onto the 10-position of 1 via the coupling agent
dicyclohexylcarbodiimide (DCC).16 For easier workup in
the preparation of 3n , ethyl N,N-dimethylaminopropyl-
carbodiimide (EDC) was used in place of DCC. EDC was
also used to obtain the hydroxamate 3o from 3n and
N-methylhydroxylamine hydrochloride. For the prepa-
ration of 3q, the amino group of 4-aminophenylacetic
acid was protected as the tert-butoxycarbonyl (BOC)
derivative 2p . Reaction of 2p with 1 in the presence of
DCC produced analogue 3p , which upon deprotection
with trifluoroacetic acid gave 3q.
The other starting arylacetic acids were either com-
mercial products or prepared according to the literature.
Scheme 2 shows the preparation of 4-ethoxycarbonyl
analogue 2c (for the preparation of 3c), which was
obtained from the appropriate ethyl phenylacetate by
selective saponification of the aliphatic ester group by
analogy to the method of Maillard.17 Benzyloxyphenyl-
acetic acid 2ii (for the preparation of 3ii) was produced
from methyl 4-hydroxyphenylacetate by reaction with
Results from the HaCaT keratinocyte proliferation
assay show that the phenylacetyl analogue 3a also
retains the antiproliferative activity of the parent 1,
although the potency is somewhat reduced. Compounds
3b-m compare the effects of a number of electron-
withdrawing groups at the para-position. It can be seen
that, compared with hydrogen, no marked improve-
ments are obtained. Although the para-bromo derivative
3k is a potent inhibitor of LTB4 biosynthesis, inhibition
against HaCaT cell growth is decreased as compared
to 1 and 3a .

816 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Mu¨ller et al.
Ta ble 1. Deoxyribose Degradation, Antiproliferative Activity, and Cytotoxicity against HaCaT Cells and Inhibition of LTB4
Biosynthesis in Bovine PMNL by 10-Arylacetyl-1,8-dihydroxy-9(10H)-anthracenones
compd
R1
R2
DD (•OH)a
<0.3
<0.3
<0.3
ND
<0.3
ND
<0.3
<0.3
ND
AA IC50 (µM)b
LDH (mU)c
LTB4 IC50 (µM)d
3a e
3b
3c
3d
3ee
3f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.9
1.8
1.3
3.2
4.7
1.6
2.6
2.2
1.2
2.9
3.1
2.2
1.7
0.7
1.8
1.2
1.3
1.7
3.0
1.6
0.7
1.1
1.6
2.7
1.9
4.2
3.3
1.7
1.6
1.8
1.2
2.4
3.2
1.0
1.3
1.0
3.5
0.9
1.1
ND
164
ND
ND
179
ND
ND
ND
ND
ND
ND
ND
ND
199
ND
ND
ND
173
199
ND
145g
143g
125g
ND
ND
ND
ND
142g
132g
ND
ND
202
ND
159
247
ND
ND
191
ND
11
12
6
9
7
11
11
1
4-CO2Me
4-CO2Et
4-CN
4-NO2
4-CF3
4-F
4-Cl
3,4-Cl2
4-Br
4-I
4-Ph
3g
3h
3i
3k
3l
9
<0.3
<0.3
<0.3
0.7
1.5
7
3
0.05
2
3m
3n
3o
3p
3q
3r
4-CH2COOH
4-CH2CON(Me)OH
4-NHBOC
4-NH2
0.93 ( 0.09f
0.51 ( 0.08f
0.97 ( 0.13f
1.43 ( 0.10f
1
6
4-NMe2
4-OH
ND
3se
3te
3u
3v
0.36 ( 0.07f
14
10
6
0.6
0.5
17
3
8
22
1
3,5-(t-Bu)2-4-OH
4-SMe
<0.3
ND
4-Me
4-OMe
2-OMe
3-OMe
<0.3
<0.3
<0.3
<0.3
ND
3w e
3x
3y
3z
3-OH-4-OMe
3,4-(OMe)2
3,4-OCH2O
4-OEt
3a a
3bb
3cc
3d d
3ee
3ff
0.86 ( 0.20f
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
<0.3
1
1
4-OPr
4-O-i-Pr
4-OBu
15
1.5
0.6
1.5
1
3gge
3h h
3ii
3k k
3ll
3m m
3n n
3oo
4-OBn
H
H
H
4-O(4-MeOBn)
4-O(3-MeOBn)
2-NH(Ph-2,6-Cl2)
H
0.45 ( 0.01f
3
Meh
Meh
Meh
Ph
<0.3
<0.3
<0.3
<0.3
18
16
9
4-i-Bu
4-Bz
H
7
Ar
3p p
3qq
3r r
3ss
1-naphthyl
2-naphthyl
cyclohexyl
3-indolyl
<0.3
<0.3
<0.3
<0.3
ND
2.6
2.9
1.7
0.8
3.9
ND
ND
ND
174
ND
37
3
3
13
9
3tt
3-indolyl of indomethacin
15
anthralin (1)
2.89 ( 0.14f
0.7
294
a
Deoxyribose degradation as a measure of hydroxyl-radical formation. Indicated values are µmol of malondialdehyde/mmol of deoxyribose
b
released by 75 µM test compound (controls < 0.1; values are significantly different with respect to control, P < 0.01). Antiproliferative
activity against HaCaT cells. Inhibition of cell growth was significantly different with respect to control, N ) 3, P < 0.01. c Activity of
d
LDH (mU) release in HaCaT cells after treatment with 2 µM test compound (N ) 3, SD < 10%). Inhibition of LTB4 biosynthesis in
bovine polymorphonuclear leukocytes. Inhibition was significantly different with respect to control, N ) 3 or more, P < 0.01.
Nordihydroguaiaretic acid (NDGA) was used as a standard (IC50 ) 0.4 µM). e Ref 12. f Values are significantly different with respect to
g
h
vehicle control. Values are not significantly different with respect to vehicle control. Racemate. ND ) not determined.
Preliminary experiments indicated that addition of a
para-methoxy group (3w ) to the unsubstituted 3a
greatly enhanced inhibitory action against 5-lipoxyge-
nase.12 Moreover, as can be seen in Table 1, the
antiproliferative activity of 1 is retained. By contrast,
changing the methoxy group of 3w to other electron-
donating para-groups (3p -u ) is moderately disadvanta-
geous for both antiproliferative and LTB4 inhibitory
activity. The exception is the para-methyl analogue 3v
which shares the same excellent antiproliferative activ-
ity with 1 and the LTB4 inhibitory potency with 3w .
The importance of the position of the methoxy group
on the phenyl ring is investigated by comparing ana-
logues 3w -y. Moving the methoxy group to the ortho-

Antipsoriatic Anthrones with Modulated Redox Properties
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 817
or meta-position as in 3x and 3y results in a 34- and
6-fold decrease in inhibition of LTB4 biosynthesis,
respectively. Also, inhibition of keratinocyte growth is
decreased. If, however, the methoxy group is moved
away from the phenylacetyl group by insertion of a
benzyl group, slightly better potency is observed for the
meta-methoxy analogue 3ii as compared to the para-
methoxy analogue 3h h .
states including psoriasis.28 Accordingly, inhibition of
the enzymes or antagonism of leukotriene biosynthesis
is explored as a potentially viable approach to anti-
psoriatic therapy.29-31 An antipsoriatic agent that in-
hibits the biosynthesis of LTB4 in addition to growth-
inhibitory effects on keratinocytes may reflect improved
antipsoriatic activity with respect to the inflammatory
component of psoriasis.
LTB4 can also effect keratinocyte proliferation,32,33
and cultured HaCaT keratinocytes express the 5-lipoxy-
genase gene.34 Consequently, the better antiproliferative
activity observed with compounds 3o and 3v may be
related to the more potent LTB4 inhibitory activity also
seen with those compounds. However, based upon the
weaker results obtained with the potent LTB4 biosyn-
thesis inhibitors 3k and 3gg in the keratinocyte assay,
it is unclear whether inhibition of LTB4 biosynthesis
actually plays a role in the antiproliferative activity of
these compounds. Therefore, the antiproliferative activ-
ity of the para-substituted analogues and their ability
to inhibit LTB4 biosynthesis was compared. Plots of the
log IC50 for inhibition of HaCaT cell growth versus log
IC50 for inhibition of LTB4 biosynthesis (data not shown)
do not support any relationship between the effects of
the compounds in the two kinds of assays (r ) 0.06).
Mem br a n e Da m a ge. Furthermore, keratinocytes
were also tested for their susceptibility for the action of
the compounds on plasma membrane integrity. Cyto-
toxicity of the cell cultures was assessed by the activity
of lactate dehydrogenase (LDH) released into the culture
medium,19 which is commonly used as an indicator of
plasma membrane damage. As a result of its potential
to generate oxygen radicals, parent 1 is an inducer of
lipid peroxidation in biological membranes.35 In contrast
to 1, which shows a 2-fold increase in LDH activity as
compared to controls, LDH release after treatment of
HaCaT cells with the most potent inhibitors of cell
growth, 3o and 3v, is markedly decreased or does not
exceed the control values, respectively.
Analogues 3z and 3a a explore the effects of an
additional hydroxyl or an extra methoxy group, respec-
tively. In both cases, potency is decreased as compared
to 3w . Incorporating the methoxy group in the cyclic
3,4-methylenedioxy substituent (3bb), which maintains
the electronic character of the acyclic analogue 3a a but
introduces additional structural rigidity, gives satisfac-
tory potency only in the LTB4 assay.
The series 3w ,cc-ff further explores the utility of
4-alkoxy substitution, covering alkyl groups from methyl
to butyl. No further increase in potency is seen at longer
chain length (3cc,d d ,ff) or a branched chain (3ee).
While potent inhibition of LTB4 biosynthesis is main-
tained with a benzyloxy group (3gg), growth-inhibitory
effects are decreased.
Furthermore, incorporation of arylacyl moieties of
antiinflammatory agents such as diclofenac (3k k ), ibu-
profen (3m m ), ketoprofen (3n n ), and indomethacin (3tt)
into hybridized structures decreases LTB4 inhibitory
action as compared with that of the most active com-
pounds (3k ,o,v,w ,gg) of this series. However, except for
the lipophilic ibuprofen and indomethacin derivatives,
potent inhibition of keratinocyte growth is observed.
Since the potential of generating potent inhibitors of
leukotriene biosynthesis by incorporating a hydroxam-
ate moiety has been described,21,22 and in particular
N-methylhydroxamate proved to be useful in 2-substi-
tuted anthracenones,23 we also explored the utility of
such a functional group. Indeed, substitution of the
para-position of the phenyl ring with an N-hydroxy-N-
methylacetamide group (3o) resulted in the most potent
inhibitor of LTB4 biosynthesis within the entire series
of antipsoriatic anthracenones described so far. With an
IC50 value in the nanomolar range, the potency of 3o is
comparable to those of the most potent leukotriene
biosynthesis inhibitors.24 In addition, this analogue
retains the high antiproliferative activity of the parent
1.
Finally, we were also interested in seeing if replace-
ment of the terminal phenyl ring of the C-10 substituent
with a nonaromatic, annelated, or heterocyclic ring led
to active compounds. With respect to the unsubstituted
phenylacetyl analogue (3a ), replacement with a naph-
thalene system (3p p ,qq) or cyclohexane ring (3r r ) does
not result in dramatic changes in activity, whereas the
antiproliferative activity of the 3-indole analogue 3ss
is significantly increased.
Among the variety of diseases in which the leuko-
trienes, the products of the 5-lipoxygenase pathway of
arachidonic acid metabolism, play a symptomatic or
causative role is psoriasis.25 LTB4 regulates accumula-
tion of polymorphonuclear leukocytes,26 and application
of LTB4 induces changes similar to those found in
psoriasis.27 Although still controversial,24 there is sub-
stantial evidence that LTB4 may play a significant role
in the amplification of many inflammatory disease
In d u ction of Ker a tin ocyte Differ en tia tion . Be-
sides increased inflammation of the skin and keratino-
cyte hyperproliferation, abnormal keratinocyte differ-
entiation is an important pathogenic factor of psoriasis.2
Since differentiation is an irreversible process, killing
aberrant cells which are then lost by sloughing, agents
that induce this process can be used in the treatment
of hyperproliferative conditions. HaCaT cells, although
immortalized and genetically abnormal, share many
features of differentiation with normal keratinocytes,36
and the highly preserved epidermal characteristics
make this cell line an excellent candidate for studying
external modulators of epidermal differentiation.37 Ke-
ratinocytes change in cell morphology, increase in cell
size, and change in the proportion of cells that increase
the amount of cornified envelope protein, a major
structural component generated as a result of the
process of keratinocyte differentiation.38 The cornified
envelopes are composed predominantly of an array of
cross-linked proteins.39 To further investigate the anti-
psoriatic potential of the novel anthracenones, we
examined their ability to induce the formation of cross-
linked protein envelopes. The data of representative
anthracenones are presented in Table 2.

818 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Mu¨ller et al.
Ta ble 2. Induction of Keratinocyte Differentiation by Selected
Anthracenones and Standard Drugs
withdrawal of cells from the cell cycle, which is many
times faster in psoriasis than normal.2
cross-linked envelope assaya
In conclusion, the present study has confirmed that
arylacetyl substituents at the 10-position of 1 provide
analogues with diminished hydroxyl-radical formation
and improved inhibitory action against LTB4 biosyn-
thesis. Furthermore, appropriate para-substitution in
the 10-phenylacetyl side chain can modify 1 such that
it retains potent antiproliferative activity combined with
reduced membrane damage and the ability to induce
terminal differentiation of keratinocytes. However, com-
parison of the ability of the para-substituted analogues
to inhibit LTB4 biosynthesis and their ability to arrest
keratinocyte growth does not support any relationship
between the effects of the analogues in the two kinds of
assays. The most potent analogues, 3o and 3v, show
an attractive profile of biological activity and compare
favorably with 1 as potential alternatives for anti-
psoriatic therapy.
(µg cross-linked protein/mg protein)
compd
1 µM
5 µM
10 µM
3g
3m
3o
3q
3s
0.6 ( 0.2
1.8 ( 0.6
2.8 ( 0.2b
0.0 ( 1.9
2.5 ( 0.9
2.1 ( 0.7
2.3 ( 0.3b
2.5 ( 0.2b
1.8 ( 0.4
3.7 ( 1.8b
0.6 ( 1.8
0.5 ( 1.2
-1.0 ( 0.3
2.4 ( 0.3b
3.8 ( 0.5b
5.5 ( 0.2b
6.0 ( 0.3b
5.1 ( 0.3b
4.2 ( 0.4b
5.3 ( 0.2b
3.4 ( 0.4b
4.9 ( 0.3b
4.9 ( 0.8b
-0.1 ( 0.7
0.3 ( 0.6
3.5 ( 0.1b
4.6 ( 0.5b
6.2 ( 0.5b
6.6 ( 0.3b
6.2 ( 0.2b
5.1 ( 0.1b
5.8 ( 0.5b
3.4 ( 0.2b
6.2 ( 0.4b
5.8 ( 1.4b
0.0 ( 1.0
0.8 ( 1.8
0.9 ( 0.6
3v
3w
3ff
3ss
anthralin (1)
danthron
9(10H)-anthracenone
10-Bn-anthralin
-0.8 ( 0.1
a
Differences of the amounts of cross-linked protein as
a
measure of HaCaT keratinocyte differentiation at indicated con-
centrations of test compounds and vehicle control. Results are the
b
means ( SEM of three independent experiments. Values are
significantly different with respect to vehicle control (P < 0.05,
Student’s t-test).
Exp er im en ta l Section
Melting points were determined with a Bu¨chi 510 melting
point apparatus and are uncorrected. Lipophilicities, as indi-
cated by log P values, were determined by a standard HPLC
procedure.43 Chromatography refers to column chromatogra-
phy on silica gel (E. Merck, 70-230 mesh); eluants are given
Anthralin is able to induce cross-linked protein as a
measure of terminal differentiation in the keratinocyte
cell line HaCaT in a concentration-dependent manner.
By contrast, the unsubstituted 9(10H)-anthracenone
and the anthralin metabolite danthron (1,8-dihydroxy-
9,10-anthracenedione), which are both therapeutically
inactive and do not arrest keratinocyte growth,11 are not
able to induce keratinocyte differentiation. Also, alkyl
substitution in the 10-position of anthralin as in 10-
benzylanthralin12 does not exhibit appreciable activity
within the concentration range, indicating that struc-
tural features necessary for cross-linking do not reside
necessarily in the anthralin chromophore. On the other
hand, the novel 10-arylacetyl analogues are all able to
induce significant cross-linking, with compounds 3o,
3w , and 3ff doing so down to the lowest concentration
of 1 µM. These compounds also effect growth arrest at
concentrations required to induce protein cross-linking,
demonstrating a causality between these two effects. In
contrast to parent 1 which induces membrane damage,
nonspecific toxicity cannot explain the changes caused
by the novel anthracenones insofar as treated kerati-
nocytes had normal membrane function (assessed by
LDH release) and synthesized protein faster than
control cells. While for other 10-arylacetyl analogues
growth inhibition may occur at slightly lower concentra-
tions than those required to induce differentiation,
comparatively small differences in cross-linking potency
at the concentrations used make a consistent SAR
correlation within this series of analogues difficult to
derive. A somewhat higher cross-linking potency is
generally found with more hydrophilic compounds (log
P values are given in Table 3).
1
in Table 3. H NMR spectra were recorded with a Varian EM
390 (90 MHz) or a Bruker Spectrospin WM 250 spectrometer
(250 MHz), using tetramethylsilane as an internal standard.
Fourier transform IR spectra (KBr) were recorded on a Nicolet
510M FTIR spectrometer. UV spectra were recorded on a
Kontron 810 spectrometer. Mass spectra (EI, unless otherwise
stated) were obtained on a Varian MAT CH5 spectrometer (70
eV). HPLC (Kontron 420, 735 LC UV detector) was performed
on a 250- × 4-mm column (4- × 4-mm precolumn) packed with
LiChrospher 100 RP18 (5-µm particles; Merck, Darmstadt,
Germany). Data were recorded on a MacLab data acquisition
system (WissTech, Germany) and analysis was performed with
the software Peaks on an Apple Macintosh computer.
P r ep a r a tion of P h en yla cetic Acid s. Phenylacetic acids
2 (Scheme 1, X ) OH) were commercial products or prepared
by following standard literature procedures: 2b,17 2d ,44 2p ,45
2z,46 2cc-ff,47 2h h .48
4-Eth oxyca r bon ylp h en yla cetic Acid (2c). To a solution
of Na (345 mg, 15 mmol) in absolute EtOH (40 mL) was added
ethyl 4-ethoxycarbonylphenylacetate49 (4.00 g, 16.9 mmol). The
solution was treated with absolute EtOH (100 mL) and then
with water (0.28 mL). Then it was stirred at room temperature
for 12 h, and the resulting precipitate was filtered by suction
and dissolved in water. The solution was adjusted to pH 1 with
2 N HCl, and the precipitate was extracted with EtOAc (3 ×
50 mL). The organic phase was dried over Na2SO4 and then
concentrated, and small amounts of hexane were added to
induce precipitation of white crystals: 51% yield; mp 101 °C;
FTIR 2983, 1721, 1701 cm-1; 1H NMR (DMSO-d6) δ 7.92, 7.33
(dd, J ) 9 Hz, 4H), 4.42-4.19 (q, J ) 7 Hz, 2H), 3.67 (s, 2H),
1.30 (t, J ) 7 Hz, 3H); MS m/z ) 208 (20, M+), 163 (100). Anal.
(C11H12O4) C, H.
Meth yl 4-(3-Meth oxyph en yl)m eth oxyph en ylacetate (4).
To a solution of methyl 4-hydroxyphenylacetate (3 g, 18 mmol)
in absolute acetone (50 mL) were added dry K2CO3 (13.8 g, 10
mmol), 3-methoxybenzyl chloride (4.37 g, 28 mmol), and a
catalytic amount of KI. The mixture was heated to reflux under
vigorous stirring for 12 h. Then it was filtered, the filtrate was
evaporated, and the residue was purified by chromatography
using CH2Cl2/hexane (9/1) to yield a pale yellow fluid: 60%
The results presented herein demonstrate that 10-
arylacetylanthracenones not only inhibit keratinocyte
proliferation but also induce their differentiation. While
growth arrest may be a necessary consequence of
keratinocyte differentiation,40 growth arrest does not
necessarily result in the onset of differentiation.41,42
Terminal differentiation of keratinocytes is documented
by the ability of the anthracenones to cross-link proteins
into cornified envelope-like structures, resulting in
yield; bp 201-203 °C/1600 Pa; FTIR 1736 cm-1 1H NMR
;
(CDCl3) δ 7.31-6.74 (m, 8H), 4.87 (s, 2H), 3.78 (s, 3H), 3.65
(s, 3H), 3.53 (s, 2H); MS m/z ) 286 (17, M+), 121 (100). Anal.
(C17H18O4) C, H.

Antipsoriatic Anthrones with Modulated Redox Properties
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 819
Ta ble 3. Chemical Data of 10-Arylacetyl-1,8-dihydroxy-9(10H)-anthracenones
compd
log Pa
formulab
C24H18O6
methodc
mp (°C)
% yieldd
solvente
3b
3c
3d
3f
3g
3h
3i
3k
3l
3m
3n
3o
3p
3q
3r
3u
3v
4.07
4.34
3.58
4.40
4.11
4.49
4.74
4.60
4.80
5.02
2.84
3.02
4.17
3.29
4.42
4.42
4.54
4.20
4.16
3.41
3.77
4.06
4.51
4.97
4.59
5.41
4.91
5.19
3.91
4.43
5.57
4.46
4.60
4.61
4.71
4.97
3.77
5.17
A
A
B
B
A
A
B
A
A
B
C
c
197-198
161-162
188-190f
165-167
178-180
163-165
162-163
179-181
190-191
188f
20
18
36
29
39
34
26
30
34
22
16
5
38
67
31
28
18
31
21
26
23
25
21
25
24
34
24
28
6
MC/E (24/1)
MC/E (9/1)
MC/E (9/1)g
MC/H (8/2)h
MC
C
C
25H20O6
23H15NO4
C23H15F3O4
C22H15FO4
C
22H15ClO4
MC/H (8/2)i
MC/PE (7/3)j
MC/H (8/2)i
MC/H (8/2)
MC/PE (7/3)k
MC/M (9/1)g
MC/M (98/2)
MC/E (19/1)
l
C22H14Cl2O4
C22H15BrO4
C22H15IO4
C28H20O4
C24H18O6
197f
C25H21NO6
150f
C
C
C
27H25NO6
22H17NO4
24H21NO4
B
c
204-206f
163-164
178f
B
B
B
A
A
B
A
A
A
A
A
A
A
A
B
A
B
B
B
B
B
B
B
B
MC/EE (99/1)k
i
C23H18O4S
23H18O4
C23H18O5
167f
C
152-154
178-179
140
138-141
165-166
182
162
135-136
143
MC/H (8/2)h
MC/H (17/3)
MC/H (17/3)
MC/E (9/1)i
MC/E (19/1)
MC/H (7/3)
MC/H (8/2)
MC/H (17/3)
MC
3x
3y
3z
C
C
23H18O5
23H18O6
3a a
3bb
3cc
3d d
3ee
3ff
3h h
3ii
3k k
3ll
3m m
3n n
3oo
3p p
3qq
3r r
3ss
3tt
C24H20O6
C
C
C
C
C
C
C
C
23H16O6
24H20O5
25H22O5
25H22O5
26H24O5
30H24O6
30H24O6
28H19Cl2NO4
114
159
MC/H (9/1)
MC/H (8/2)
MC/H (19/1)
MC/PE (7/3)m
MC/H (7/3)
MC/PE (6/4)n
MCo
147-149
183-184
184-185
179-181
128-133
193f
C23H18O4
C27H26O4
C30H22O5
C28H20O4
C26H18O4
C26H18O4
C22H22O4
C24H17NO4
C33H24ClNO6
13
9
9
13
20
20
21
28
10
MC/PE (1/1)j
MC/PE (1/1)j
MC/PE (6/4)k
EE/PE (3/7)j
MCo
207f
159-162
139-142
193f
148-150
MCo
a
b
Experimentally determined partition coefficients.43 All new compounds displayed 1H NMR, FTIR, UV, and MS spectra consistent
with the assigned structure. Elemental analyses were within (0.4% of calculated values. c See Experimental Section. Yields have not
d
been optimized. e Chromatography solvent (vol %): A ) acetic acid; E ) ether; EE ) ethyl acetate; H ) hexane; M ) methanol; MC )
methylene chloride; PE ) petroleum ether. f Decomposition. g Recrystallized from benzene/THF (8/2). Recrystallized from benzene/hexane
h
i
j
k
(7/3). Recrystallized from benzene. Recrystallized from benzene/petroleum ether (1/1). Recrystallized from benzene/petroleum ether
l
m
n
(6/4). Recrystallized from benzene/ethanol (8/2). Recrystallized from methylene chloride/petroleum ether (1/1). Recrystallized from
benzene/petroleum ether (2/8). o Recrystallized from benzene/petroleum ether (8/2).
4-(3-Meth oxyp h en yl)m eth oxyp h en yla cetic Acid (2ii).
A suspension of 4 (2.29 g, 8 mmol) in 6 N NaOH (30 mL) was
heated to reflux for 4 h. The mixture was cooled and neutral-
ized with 6 N HCl, and the resulting precipitate was extracted
with EtOAc (3 × 30 mL). The organic phase was dried over
Na2SO4 and then concentrated. Petroleum ether (40-60) was
added to induce precipitation of white crystals: 66% yield; mp
86 °C; FTIR 1717 cm-1; 1H NMR (DMSO-d6) δ 12.24 (br s, 1H),
7.40-6.82 (m, 8H), 4.89 (s, 2H), 3.75 (s, 3H), 3.49 (s, 2H); MS
m/z ) 272 (17, M+), 121 (100). Anal. (C16H16O4) C, H.
P r ep a r a tion of P h en yla cetyl Ch lor id es. Phenylacetyl
chlorides 2 (Scheme 1, X ) Cl) were prepared from the
corresponding phenylacetic acids by following standard lit-
erature procedures.14,15 In most cases the crude products were
used in the subsequent acylation steps.
(CDCl3) δ 12.27 (2H), 7.55-6.58 (m, 10H), 5.30 (s, 1H), 3.27
(s, 2H); MS m/z 424 (3.5), 422 (3.8). Anal. (C22H15BrO4) C, H.
Met h od B. 1,8-Dih yd r oxy-10-[2-(4-m et h ylp h en yl)-1-
oxoeth yl]-9(10H)-a n th r a cen on e (3v). To a solution of an-
thralin50 (1; 1.00 g, 4.42 mmol), p-tolylacetic acid (2v, X ) OH;
0.80 g, 5.30 mmol), and DCC (1.10 g, 5.30 mmol) in absolute
THF (30 mL) was added dry pyridine (2.0 mL) under N2. The
reaction mixture was stirred at room temperature for 4 h,
filtered, and the filtrate was evaporated. The residue was
purified by chromatography and recrystallized from benzene/
hexane (7/3) to afford 3v as yellow needles (Table 3): FTIR
1711 (COOH), 1630 cm-1 (CO‚‚‚HO); 1H NMR (CDCl3) δ 12.26
(2H), 7.54-6.59 (m, 10H), 5.31 (s, 1H), 3.33 (s, 2H), 2.26 (s,
3H). Anal. (C23H18O4) C, H.
Meth od C. 4-[2-(4,5-Dih yd r oxy-10-oxo-9,10-d ih yd r oa n -
th r a cen -9-yl)-2-oxoeth yl]p h en yla cetic Acid (3n ). To a
solution of anthralin50 (1; 4.85 g, 21.5 mmol), 1,4-phenylene-
diacetic acid (2n , X ) OH; 4.17 g, 21.5 mmol), and EDC (8.24
g, 43 mmol) in absolute THF (200 mL) was added dry pyridine
(3.0 mL) under N2. The reaction mixture was stirred for 2 h
at 50 °C and an additional 12 h at room temperature. Then it
was treated with 2 M HCl (200 mL), and the mixture was
extracted with EtOAc (3 × 100 mL). The combined organic
phase was washed with water, dried over Na2SO4, and the
solvent was evaporated. The residue was purified by chroma-
tography and recrystallized from benzene/THF (8/2) to afford
a pale yellow powder (Table 3): FTIR 1736 (COOH), 1707
Gen er a l P r oced u r e for th e P r ep a r a tion of 10-Ar yl-
a cetyl-1,8-d ih yd r oxy-9(10H)-a n th r a cen on es. Meth od A.
10-[2-(4-Br om op h en yl)-1-oxoeth yl]-1,8-d ih yd r oxy-9(10H)-
a n th r a cen on e (3k ). To a solution of anthralin50 (1; 1.00 g,
4.42 mmol) in absolute THF (30 mL) and dry pyridine (1.0 mL,
12.70 mmol) was added dropwise a solution of 4-bromophen-
ylacetyl chloride (2k , X ) Cl; 1.03 g, 4.42 mmol) in absolute
THF (10 mL) under N2. The reaction mixture was stirred at
room temperature for 4 h, filtered, and the filtrate was
evaporated. The residue was purified by chromatography and
recrystallized from benzene to afford 3k as yellow needles
(Table 3): FTIR 1711 (COOH), 1630 cm-1 (CO‚‚‚HO); 1H NMR

820 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Mu¨ller et al.
(CO), 1628 cm-1 (CO‚‚‚HO); 1H NMR (DMSO-d6) δ 12.50-11.50
(br, 1H), 11.93 (s, 2H), 7.64-6.90 (m, 10H), 5.75 (s, 1H), 3.96
(s, 2H), 3.51 (s, 2H); MS m/z 402 (2.5), 226 (100). Anal.
(C24H18O6) C, H.
(2) McKay, I. A.; Leigh, I. M. Altered keratinocyte growth and
differentiation in psoriasis. Clin. Dermatol. 1995, 13, 105-114.
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J . Clin. Pharm. Ther. 1994, 19, 223-232.
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3a ,e,s,t,w ,gg were prepared as described.12
830.
(5) Shuster, S. 1. Dithranol and psoriasis - Why do we settle for
second best? 2. Aging & retinoids, who’s fooling who? J . Eur.
Acad. Dermatol. Venereol. 1997, 9, S99.
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Dermatol. Venereol. 1997, 9, S33.
(7) Harris, D. R. Old wine in new bottles: the revival of anthralin.
Cutis 1998, 62, 201-203.
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cenone antipsoriatic agents. Curr. Pharm. Des. 2000, 6, 1041-
1057.
(9) Mu¨ller, K. Antipsoriatic and proinflammatory action of anthra-
lin. Implications for the role of oxygen radicals. Biochem.
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(10) Lange, R. W.; Hayden, P. J .; Chignell, C. F.; Luster, M. I.
Anthralin stimulates keratinocyte-derived proinflammatory cy-
tokines via generation of reactive oxygen species. Inflamm. Res.
1998, 47, 174-181.
N-Hyd r oxy-N-m eth yl-4-[2-(4,5-d ih yd r oxy-10-oxo-9,10-
dih ydr oan th r acen -9-yl)-2-oxoeth yl]ph en ylacetam ide (3o).
A solution of 3n (1.00 g, 2.49 mmol), N-methyl-N-hydroxyl-
amine (0.42 g, 4.97 mmol), and EDC (0.95 g, 4.97 mmol) in
absolute DMF (20 mL) was stirred at room temperature for 5
h under N2. Then cold 2 M HCl (100 mL) was added, the
precipitate was filtered by suction, washed with water (100
mL), and dried under vacuum. The residue was treated with
THF (100 mL), dried over Na2SO4, and the solvent was
evaporated. The residues were purified by chromatography to
give a yellow-brown powder (Table 3): FTIR 3417 (OH), 1713
1
(CO), 1630 cm-1 (CO‚‚‚HO); H NMR (DMSO-d6) δ 11.93 (s,
2H), 9.96 (s, 1H), 7.65-6.88 (m, 10H), 5.75 (s, 1H), 3.95 (s,
2H), 3.64 (s, 2H), 3.09 (s, 3H); LSIMS (3-nitrobenzylacohol/
CH2Cl2) m/z 431.4 (M+). Anal. (C25H21NO6) C, H.
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analogues of anthralin: unusual specificity of structure and
antiproliferative activity. J . Med. Chem. 1997, 40, 3773-3780.
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anthrones with modulated redox properties. 1. Novel 10-
substituted 1,8-dihydroxy-9(10H)-anthracenones as inhibitors of
5-lipoxygenase. J . Med. Chem. 1993, 36, 4099-4107.
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Loewengart, G.; Mate´, U.; Roth, D.; Smith, A.; Melchionne, S.;
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Biologica l Assa y Meth od s. Deoxyribose degradation as
a measure of hydroxyl-radical generation,12 HaCaT kerati-
nocyte proliferation assay,18 LDH release as a measure of
membrane damage,19 and inhibition of LTB4 biosynthesis in
bovine polymorphonuclear leukocytes12 were described previ-
ously in full detail.
Cor n ified En velop e Assa y. The cornified envelope is a
well-recognized marker of terminal differentiation in kerati-
nocytes.38 The assay measures insoluble cross-linked protein
envelopes based on previous reports.39,51 HaCaT cells were
cultured as described.18 The medium of 144-h post-confluent
cultures was replaced, and the test compounds were added
from stock solutions (1, 5, 10 µM). These were prepared in
DMSO and then diluted with Dulbecco’s modified Eagle’s
medium; the final concentration of DMSO was 0.2% in the
culture medium. Controls were performed with DMSO or
medium alone. 48 h after addition of the test compounds to
the culture, the medium was removed and each well was
rinsed with phosphate-buffered saline (PBS; 100 µL). The cells
were then incubated with sterile 0.5% trypsin, 0.2% EDTA in
PBS for 20 min at 37 °C. The detached cells from each well
were treated with 10% sodium dodecyl sulfate and 2% â-mer-
captoethanol (SDS/âME) in water (100 µL) with vigorous
agitation. A sample (250 mL) of the suspension was applied
to a presoaked, regenerated cellulose sheet (RC 60, Schleicher
and Schuell, Dassel, Germany) over a dot-blot apparatus (Bio-
Dot, BIO-RAD, Munich, Germany) using a presoaked, protein-
free cellulose sheet (GB 002, Schleicher and Schuell) as a
backing. The suspension was drained by gentle suction from
below, and wells were washed with SDS/âME (3 × 400 µL).
The RC 60 sheet was dried and then removed from the
apparatus, submerged in ice-cold 7.5% TCA solution (500 mL),
heated to 80 °C for 30 min, washed with ether/EtOH (1/1) for
10 min and then with ether (250 mL) for 10 min. The RC 60
sheet was dried and stained with a solution of 1% Coomassie
blue G-250 in 7% acetic acid (500 mL) at 50 °C for 15 min.
Then it was washed with 7% acetic acid at 50 °C for 5 min.
This was repeated until the background was white (usually
two times). The sheet was then transferred to a scanner
(Hewlett-Packard Scan J et 4c), and image analysis was
performed with Optimas 6.1 (Media Cybernetics, Go¨ttingen,
Germany) on a Pentium Vectra computer. Indicated values
are the differences of the amount of cross-linked protein in
the presence of test compounds and vehicle control. Protein
in envelope preparations was determined by the method of
Bradford.52
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C.; Lannoy, J .; Morin, R.; Lefebvre, M.-J .; Manuel, C.; Verro,
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a
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