Antipsoriatic anthrones with modulated redox properties. 3. 10-Thio- substituted 1,8-dihydroxy-9(10H)-anthracenones as inhibitors of keratinocyte growth, 5-lipoxygenase, and the formation of 12(S)-HETE in mouse epidermis

Article abstract of DOI:10.1021/jm960259l

The synthesis of a series of 1,8-dihydroxy-9(10H)-anthracenones bearing sulfur-linked substituents in the 10-position is described. These compounds were evaluated for their ability to inhibit the growth of the human keratinocyte cell line HaCaT and the 5-

Full text of DOI:10.1021/jm960259l

3132
J . Med. Chem. 1996, 39, 3132-3138
An tip sor ia tic An th r on es w ith Mod u la ted Red ox P r op er ties. 3.
10-Th io-Su bstitu ted 1,8-Dih yd r oxy-9(10H)-a n th r a cen on es a s In h ibitor s of
Ker a tin ocyte Gr ow th , 5-Lip oxygen a se, a n d th e F or m a tion of 12(S)-HETE in
Mou se Ep id er m is1
Klaus Mu¨ller,*,† Hsu-Shan Huang,‡ and Wolfgang Wiegrebe†
Institut fu¨r Pharmazie, Pharmazeutische Chemie I, Universita¨t Regensburg, D-93040 Regensburg, Germany, and
Institute of Pharmacy, National Defense Medical Center, Taipei, Taiwan, R.O.C.
Received April 3, 1996X
The synthesis of a series of 1,8-dihydroxy-9(10H)-anthracenones bearing sulfur-linked sub-
stituents in the 10-position is described. These compounds were evaluated for their ability to
inhibit the growth of the human keratinocyte cell line HaCaT and the 5- and 12-lipoxygenase
enzymes in bovine polymorphonuclear leukocytes and mouse epidermal homogenate, respec-
tively. In addition, the following redox properties of the compounds were determined: reactivity
against 2,2-diphenyl-1-picrylhydrazyl, generation of hydroxyl radicals as measured by deoxy-
ribose degradation, and inhibition of lipid peroxidation in model membranes. Compounds 4e
and 4h of this series compare favorably in the cellular assays with the antipsoriatic anthralin.
They have the combined inhibitory action against leukotriene B4 and 12(S)-HETE formation
and are highly potent antiproliferative agents against keratinocyte growth. In contrast to
anthralin, 4h , 1,8-dihydroxy-10-[(4-hydroxyphenyl)thio]-9(10H)-anthracenone, is not cytotoxic
as documented by the LDH activity released from cytoplasm of keratinocytes and does not
enhance lipid peroxidation in model membranes.
Ch a r t 1
Anthralin (1) is among the most widely used drugs
in the treatment of psoriasis.2 However, its clinical
efficacy is limited by irritation of the nonaffected skin
surrounding the psoriatic lesion and its staining of skin
and clothing. These are significant drawbacks which
are serious enough to prevent its use or reduce patient
compliance.3 Accordingly, the development of a topically
effective antipsoriatic agent which should obviate the
undesired side effects is highly desirable.4,5 The mech-
anism of anthralin-induced skin irritation is not com-
pletely understood, but several studies have suggested
that it may be associated in part with the formation of
active oxygen species6,7 or anthralin radicals8,9 and
subsequent lipid peroxidation.7,10 Our strategy for
overcoming these problems was to modulate the radical-
generating intensity of anthralin and stabilize the 1,8-
dihydroxy-9(10H)-anthracenone pharmacophore by modi-
fying the critical 10-position of the molecule.
inhibitory and antiproliferative activities. This paper
describes the design and synthesis of anthracenones
that incorporate in their structure a potential antioxi-
dant component (thioether side chain) and the results
of relevant biological studies.
In a previous paper, we described the synthesis and
structure-activity relationships for 10-acyl derivatives
of anthralin.11 This modification of anthralin, which
gave rise to compounds of the general formula 2,
enhanced the activity against 5-lipoxygenase (LO) by
two orders of magnitude. The compounds of this series
appear to inhibit 5-LO by specific enzyme interaction
rather than nonspecific redox inhibition. Currently,
clinical trials in psoriasis are underway with 2a .
In order to provide further insight into the anthra-
cenone pharmacophore and the involvement of free
radicals, we examined the effects of introducing electron-
donating 10-thio substituents to see whether replace-
ment of the electron-withdrawing carbonyl of the earlier
series can provide analogs with both potent LO enzyme
Ch em istr y
Ap p lica tion of th e Ca p tod a tive Effect. For the
1,8-dihydroxy-9(10H)-anthracenone-10-yl radical, two
mesomeric forms 1R,â (Scheme 1) account for the
conjugative stabilization effect by the 9-keto function,
an acceptor group at a vinylogous position. It has been
postulated that the stability of free radicals is enhanced
by the combined action of an electron-withdrawing and
an electron-donating group attached to the same or to
two vinylogous carbon atoms.12 Accordingly, a donor
substituent by its nature at the 10-position of the
anthracenone nucleus contributes a stronger stabiliza-
tion to the electrophilic radical 1R,â than an acceptor
substituent, a type of resonance stabilization called the
captodative effect or push-pull substitution.12,13 On the
basis of this concept, a 10-thio group was assumed a
suitable substituent to fulfill these requirements. In
* To whom correspondence should be addressed.
† Universita¨t Regensburg.
‡ National Defense Medical Center
X Abstract published in Advance ACS Abstracts, J uly 15, 1996.
S0022-2623(96)00259-2 CCC: $12.00 © 1996 American Chemical Society

Antipsoriatic Anthrones with Modulated Redox Properties
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3133
Sch em e 1. Mesomeric Forms of Anthralin Radical 1R,â
and Stabilization by an Electron-Withdrawing Keto
Substituent (2R-γ) and an Electron-Donating Thio
Substituent (3R-ꢀ)
Sch em e 2a
a
R is defined in Table 1. Reagents: (a) Br2, CS2, 50 °C; (b) RSH,
TFA, CH2Cl2, room temperature.
which strongly reduced the reaction time as compared
to the noncatalyzed reaction. Compounds containing an
arylthio or aralkylthio substituent (4) at C-10 were
obtained according to the same procedure.
Biologica l Eva lu a tion
Psoriasis is a widespread, inflammatory and scaling
skin disease, which is characterized by increased cell
proliferation of the epidermis.19 Both features of pso-
riasis, hyperproliferation and inflammation, are re-
solved following topical therapy with anthralin. Al-
though there is no appropriate animal model of psoria-
sis, the biological activity of drugs useful as antipsoriatic
agents may be evaluated by their antiproliferative
activity in cell cultures,20 which may be critical in
resolving hyperproliferation of psoriasis. Furthermore,
inhibition of the biosynthesis of both leukotrienes (LT)
and hydroxyeicosatetraenoic acids (HETE) may be ap-
propriate to manage the inflammatory component of the
disease.21 On the basis of this information, we evalu-
ated the ability of the novel compounds to inhibit the
growth of human keratinocytes1 and the 5- and 12-LO
pathways in bovine polymorphonuclear leukocytes11
(PMNL) and epidermal homogenate of mice,22 respec-
tively.
An tip r olifer a tive Activity. In vitro cultured cell
systems are useful tools in identifying new topical
antipsoriatic agents. HaCaT keratinocytes can be used
as a model for highly proliferative epidermis, e.g.
psoriasis, and this nontransformed human cell line was
described as an extremely sensitive target for the
antiproliferative action of anthralin.23 Proliferation of
the keratinocytes was determined directly by counting
the dispersed cells under a phase-contrast microscope
after 48 h of treatment.
The compounds in Table 1 were tested for antiprolif-
erative effects as demonstrated by reduction in cell
number over time as compared to control plates. Except
for compounds 3a and 4l, all of them exhibited activity
against cell growth in the micromolar range. Although
there were no obvious requirements for potent antipro-
liferative activity, the (2-amino- (4e) and (4-hydroxy-
phenyl)thio (4h ) compounds were highly potent inhibi-
tors with IC50 values as low as 0.1 µM. These two
compounds were about six-fold more potent than an-
thralin, which had an IC50 of 0.6 µM in this assay.
However, only minor structural changes, such as varia-
tion of the position of the amino or hydroxy function at
the phenyl ring (4f,g) or methylation (4d ), strongly
decreased potency.
this case, two substituents of opposite polarity act on
the stabilization of the corresponding captodative radi-
cal and allow the drawing of five resonance structures
3R-ꢀ. By contrast, pairs of identical substituents act
on the radical of 10-acyl derivative 2 and only three
mesomeric forms 2R-γ are possible. The stronger
stabilization of 3 is further supported by an ESR
investigation of electronic and conformational effects in
similarly substituted phenoxyl radicals.14 Compared to
the parent phenol, the rotational barrier of a 4-meth-
ylthio-substituted phenoxyl radical was eight-fold in-
creased, whereas no significant change was observed
with a 4-acetyl group.14 This shows that the CH3S-
substituent interacts strongly with the unpaired elec-
tron and effectively withdraws the spin density from the
ring, which may not be substantial for the acetyl
substituent.14 Moreover, our preliminary ESR studies
revealed that the reduction power of anthracenones
substituted with an electron-withdrawing group in the
C-10 position is impeded, whereas that of 10-thio
derivatives is increased.15 Selected anthracenones could
be ranged according to the rate of reduction of a
nitroxide radical into the following order:15 3j > 1 > 2a .
Syn th esis. The 10-thio-substituted 1,8-dihydroxy-
9(10H)-anthracenones (3, 4) were synthesized readily
according to Scheme 2. Thus, treatment of 1 with
bromine provided 10-bromo-1,8-dihydroxy-9(10H)-an-
thracenone (5).16 In contrast to earlier reports,17,18 the
desired compounds were obtained directly from the
intermediate 5 by nucleophilic substitution at C-10 with
1.5-2 equivalents of appropriate mercaptans in the
presence of catalytic amounts of trifluoroacetic acid,
La cta te Deh yd r ogen a se Relea se. A major concern
in the testing of potential inhibitors of cell growth is to
confirm that the drug does not interfere with the
functioning of cell membrane by causing leakage of
cytoplasm through it. Anthralin has been reported to

3134 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Mu¨ller et al.
Ta ble 1. Deoxyribose Degradation, 5-LO Inhibition in Bovine PMNL, 12-LO Inhibition in Mouse Epidermis, Antiproliferative
Activity, and Cytotoxicity against HaCaT Cells by 10-Thio-Substituted 1,8-Dihydroxy-9(10H)-anthracenones
compd
R
DD (•OH)a
5-LO IC50 (µM)b
12-LO IC50 (µM)c
AA IC50 (µM)d
LDH (mU)e
3a
3b
3c
3d
3e
3f
3g
3h
3i
CH2CO2H
6.12 ( 1.02
4.89 ( 1.01
4.51 ( 0.84
4.19 ( 0.39
9.34 ( 0.33
6.34 ( 0.26
5.25 ( 0.59
5.52 ( 0.27
5.38 ( 0.75
2.33 ( 0.13
4.23 ( 0.15
2.49 ( 0.25
2.81 ( 0.78
2.45 ( 0.16
4.09 ( 0.06
4.13 ( 0.41
4.33 ( 1.02
4.29 ( 0.07
1.59 ( 0.16
4.76 ( 0.56
1.49 ( 0.32
3.70 ( 0.11
3.61 ( 0.10
3.74 ( 0.36
5.01 ( 0.02
2.78 ( 0.52
2.71 ( 0.10
2.89 ( 0.14
>30
13
>30
>30
>30
2
30(84)
>30
>5
1.9
4.5
2.1
0.2
1.9
1.1
1.6
2.3
>5
1.4
1.2
1.2
1.7
0.8
3.7
4.2
1.8
1.7
0.1
0.9
1.7
0.1
1.4
3.0
4.7
>5
0.6
ND
ND
ND
ND
ND
ND
133f
124f
ND
ND
ND
136f
117f
ND
369
ND
ND
ND
137f
255
184
174
148f
ND
ND
ND
ND
294
CH2CO2CH3
CH2CO2C2H5
CH2CO2C4H9
CH(CH3)COOH
CH2CH2CO2H
CH2CH2CO2CH3
CH2CH2CO2C2H5
CH2CH2CO2C4H9
CH2CH(NH2)COOH
CH2CH3
>30
30(66)
15
30(74)
27
15
>30
30(62)
>30
>30
2
30(69)
30(62)
30(71)
>30
3j
3k
3l
CH2CH2OH
>30
3m
3n
3o
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
CH2CH2CH3
CH2CHOHCH2OH
CH2CH2CH2SH
C6H5
2-CH3OC6H4
3-CH3OC6H4
4-CH3OC6H4
2-H2NC6H4
4-H2NC6H4
2-HOC6H4
4-HOC6H4
CH2C6H5
>30
>30
9
>30
>30
8
6
15
30(58)
30(55)
3
30(62)
30(69)
6
>30
30(73)
3
3
2
7
5
2
>30
>30
30(58)
30
30(54)
30(77)
>30
CH2C6H4OCH3
CH2CH2C6H5
CH2CH2CH2C6H5
>30
anthralin (1)
37
9
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). Inhibition of
5-HETE and LTB4 biosynthesis in bovine PMNL. c Inhibition of 12(S)-HETE biosynthesis in mouse epidermal homogenates. Inhibition
was significantly different with respect to that of the control; N ) 3 or more, P < 0.01. Values in parentheses are percent inhibition at
the indicated concentrations (µM), and standard errors average 10% of the indicated values. Nordihydroguaiaretic acid (NDGA) was
d
used as the standard inhibitor for 5- and 12-LO (IC50 ) 0.4 and 21 µM, respectively). Antiproliferative activity against HaCaT cells.
Inhibition of cell growth was significantly different with respect to that of the control, N ) 3, P < 0.01. e Activity of LDH (mU) release in
HaCaT cells after treatment with 2 µM test compound (N ) 3, SD < 10%). f Values are not significantly different with respect to vehicle
control. ND ) not determined.
cause membrane damage, but did not directly lead to
substantial membrane destruction.23 Cytotoxicity against
the cell cultures by the potent cell growth inhibitors was
assessed by the activity of lactate dehydrogenase (LDH)
activity released into the culture medium. The release
of LDH is commonly used as indicator of plasma
membrane damage. In this assay, LDH release by
anthralin significantly exceeded that of the vehicle
control. On the other hand, the activity of the novel
compounds was due to cytostatic rather than cytotoxic
effects, as LDH release was unchanged as compared to
controls. Except for thiol 3o, which shows increased
membrane damage as compared to anthralin, in all
other cases cytotoxic effects were either strongly reduced
(4e-g) or not apparent at 2 µM (3g,h ,l,m , 4d ,h )
compared to control cultures.
based oxidation,30 many LO inhibitors can be catego-
rized as antioxidants/free radical scavengers.31-35
Anthralin itself is only a moderate 5-LO inhibitor. In
isolated bovine PMNL it inhibited the production of
LTB4 and 5-HETE with an IC50 of 37 µM.11 As shown
in Table 1, we found that many compounds of the new
10-thio-substituted series were far more potent than
anthralin. We previously reported substantial increases
in 5-LO inhibitory activity by the introduction of ter-
minal phenyl rings on the 10-substituent of the anthra-
cenone.11 A similar approach within the 10-thio series
resulted also in active compounds, even though their
inhibitory activity against the 5-LO enzyme and the
beneficial effect of the terminal phenyl ring was less
pronounced as that observed with 10-phenylacyl deriva-
tives.11 Phenylthio-substituted analogs with the phenyl
ring directly attached to the sulfur linkage (4a -h ) all
demonstrated reasonable activity, whereas insertion of
methylene groups (4i-l) resulted in loss of 5-LO inhibi-
tion. Of the alkylthio-substituted compounds, only the
thiopropanoic acid 3f and thioethanol 3l exhibited
potency comparable to that of 4e or 4h . In contrast to
previous work,1,33 no correlation between 5-LO inhibi-
tion and lipophilicity of the compounds (log P values are
given in Table 3) was observed.
In h ibition of 5-Lip oxygen a se. Formation and
release of arachidonic acid metabolites from PMNL24
and keratinocytes25 participate in amplifying the in-
flammatory response. Among the most active of these
metabolites is LTB4, generated by the action of 5-LO.
Accordingly, potential antipsoriatic drugs are evaluated
for their ability to inhibit the production of LTB4.26-29
Considering that the biosynthesis of LTB4 is a radical-

Antipsoriatic Anthrones with Modulated Redox Properties
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3135
Ta ble 2. Half-lives (t1/2, h) of Selected 10-Thio-Substituted
1,8-Dihydroxy-9(10H)anthracenones in DMSO and Ethanol at
25 °C and Acidity Constants (pKa) in Aqueous Solution
In h ibition of 12(S)-HETE F or m a tion . Although
the literature of 5-LO inhibitors is vast,30 not many
compounds have been tested for 12-LO inhibitory activ-
ity. However, 12-HETE is the major LO product found
in the skin36 and has been demonstrated to be present
in high levels in psoriatic epidermis.37 Furthermore,
the characterization of LO activity in human epidermis
indicates that germinal layer keratinocytes contain a
highly active 12-LO which is selectively expressed at a
higher level during psoriatic inflammation.38 12-HETE
stimulates keratinocyte proliferation and induces his-
tological changes characteristic of psoriasis.39-41 Stud-
ies on the evaluation of 12-LO inhibitors have utilized
platelets as the source of the enzyme in most cases.42,43
Observations that potent inhibitors of the epidermal 12-
LO did not inhibit platelet derived 12-LO suggest the
existence of two distinct enzymes.44,45 Anthralin showed
higher selectivity for epidermal 12-LO versus the plate-
let enzyme (IC50 values are 9 and 30 µM, respectively).22
With psoriasis as target for therapy, potential 12-LO
inhibitors have to be evaluated using epidermal 12-
LO.21
compd
t1/2 (h) DMSO
t1/2 (h) ethanol
pKa
3i
11
28
32
26
55
23
4
2
12
21
20
34
5
5.7
5.4
7.9
8.4
6.2
6.1
ND
9.555
4a
4d
4e
4h
4j
4k
3
anthralin (1)
1857
357
M-1 s-1),11 reflecting the reducing capability of the 10-
thio substituent.
Prooxidant properties of the compounds were defined
by the deoxyribose assay.11 The release of malondial-
dehyde (MDA) is indicative of hydroxyl-radical forma-
tion. As already observed for catechol or pyrogallol
compounds of our earlier series,1,11 the potential anti-
oxidant thio component once more increased the release
of MDA (Table 1), indicating enhanced formation of
hydroxyl radicals; compounds 3j,l,n and 4d ,f were
exceptions. This is a further documentation that certain
substances can have prooxidant effects under some
reaction conditions and cannot be classified simply as
antioxidants on the basis of experiments performed with
lipid systems.51,52 Although this is somewhat disap-
pointing, however, compounds that enhance oxidative
damage to deoxyribose are not necessarily prooxidants
with respect to DNA bases.53
In this study we used epidermal strips from mouse
skin to investigate the effects of the novel 10-thio-
substituted analogs. To exclude oxidation of arachidonic
acid by a nonspecific free radical mechanism which
affords racemic 12-HETE, the absolute stereochemistry
of 12-HETE was determined by chiral phase HPLC to
confirm the presence of the S-enantiomer.22
Free radical intermediates and end-products derived
from lipid peroxidation are important components in
inflammatory diseases.54 Thus, agents that inhibit
initiation or propagation of lipid peroxidation may be
helpful in preventing tissue injury. Representative
compounds were evaluated for their efficacy to block
2,2′-azobis(2-amidinopropane) hydrochloride (AAPH)
induced lipid peroxidation in bovine brain phospholipid
liposomes11 and gave IC50 values of 24 (3c, 4a ,h ), 46
(3n ), 62 (4i,k ), and for 4l 85 µM (data not shown). By
contrast, anthralin itself was not effective against
AAPH-induced lipid peroxidation but rather enhanced
lipid peroxidation both in vivo and in model mem-
branes.7,10 Appropriate control experiments revealed
that this was not the case with the thio analogs, despite
their ability to generate hydroxyl radicals.
Sta bility. Anthralin staining is an important limita-
tion of its therapeutic use and is primarily due to its
oxidation to the anthralin radical which dimerizes or
polymerizes to give inactive products such as bianthrone
and the so-called anthralin-brown, respectively.2 With
the 10-thioether side chain, this side effect should be
reduced because of steric hindrance to radical dimer-
ization. To prove this hypothesis, we have determined
the decomposition of anthralin and some of its 10-thio
derivatives in dimethyl sulfoxide (DMSO) and ethanol
by RP-HPLC over a 10-day period at 25 °C. Table 2
shows the half-lives of selected compounds in the
corresponding solvents. In general, alkylthio substitu-
tion (3i, 4k ) decreased the stability as compared to
anthralin, whereas phenylthio-substituted compounds
(4a ,d ,e,h ) were considerably more stable. The decom-
position of the compounds was more favored in the
protic solvent ethanol than in the aprotic DMSO.
Furthermore, the acidity of the compounds may have
a considerable impact on their stability, because an-
Introduction of a 10-thio side chain containing polar
or nonpolar groups resulted in decrease in potency as
compared to anthralin, with the exception of some
phenylthio-substituted analogs. As can be seen in Table
1, (2-methoxy- (4b) and (4-hydroxyphenyl)thio substitu-
tion (4h ) at C-10 resulted in a three-fold increase in
inhibition of 12(S)-HETE formation as compared to
anthralin, and also 4e is more active than the antipso-
riatic drug. Although there was no correlation between
5- and 12-LO inhibition, compound 4h was an equipo-
tent inhibitor of both the LO enzymes.
An t ioxid a n t a n d P r ooxid a n t Det er m in a t ion .
There is no dispute that infiltration of PMNL of the
epidermis is a characteristic feature of the established
psoriatic lesion.19 Several lines of evidence suggest that
the release of oxidants by PMNL and macrophages leads
to injury to the tissue, and a similar role of active oxygen
species has been identified in psoriasis.46,47 The activity
of xanthine oxidase, an enzyme capable of generating
superoxide radical, is increased in psoriatic epidermis.48
Uncontrolled production of active oxygen species leads
to peroxidative damage to membranes of the skin, a
tissue that is particularly vulnerable to the effects of
these species.49 The increased exposure of the skin to
prooxidants together with an insufficient capacity of the
antioxidative system to respond seems to play an
important role in the induction of psoriasis by exogenous
factors.50 To counteract this oxidative stress, com-
pounds that incorporate in their structure a potential
antioxidant component and interact with free radicals
are of interest. Therefore, we determined the reactivi-
ties of the compounds with the stable free radical 2,2-
diphenyl-1-picrylhydrazyl (DPPH).11 In general, the
novel anthracenones were far more reactive (kDPPH
>
100 M-1 s-1, data not shown) than anthralin (24 ( 4.2

3136 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16
Mu¨ller et al.
Ta ble 3. Chemical Data of 10-Thio-Substituted
1,8-Dihydroxy-9(10H)-anthracenones
thralin anion is the key intermediate in the oxidation
process, which involves one-electron transfer from the
anion to oxygen to give anthralin-10-yl radical and
superoxide anion.10 Thus, absorbance spectroscopy was
employed to obtain the pKa values of the 10-substituted
derivatives of anthralin. However, there was no rela-
tionship found between stability and acidity of the
compounds. The 10-thio derivatives were all more
acidic than anthralin which had a pKa value of 9.5.55
The decomposition products of the 10-thio derivatives
in the organic solvents detected by HPLC were the
stable 1,8-dihydroxy-9,10-anthracenedione, which does
not polymerize, and the corresponding thiols, whereas
the dimer bianthrone and anthralin itself made no
contribution to the overall degradation. Thus, a phe-
nylthioether side chain at C-10 of anthralin greatly
enhances its stability in both protic and aprotic solvents
and also prevents the anthracenone molecule from being
polymerized to anthralin-brown.
compd log P
formulaa
mp, °C
yield, %
anal.b
3a
3b
3c
3d
3e
3f
3g
3h
3i
2.35
3.53
4.45
4.41
2.77
3.00
3.69
4.59
4.55
2.35
2.97
3.16
4.57
2.73
4.33
4.54
4.42
4.50
4.51
4.05
3.65
3.92
3.69
4.87
4.83
4.97
4.78
C16H12O5S
151-152c
130-131d
92-93
46
48
85
66
37
65
75
54
75
44
63
91
83
75
45
68
65
72
77
45
41
52
46
85
58
75
80
C, H
C, H
C, H
C, H
C, H
C,f H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C, H
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
17H14O5S
18H16O5S
20H20O5S
17H14O5S
17H14O5S
18H16O5S
19H18O5S
21H22O5S
90-91
168-169
147-148e
88-89g
86-87
72-73
3j
3k
3l
17H15NO5S 176-178h
16H14O3S
16H14O4S
17H16O3S
17H16O5S
17H16O3S2
20H14O3S
21H16O4S
21H16O4S
21H16O4S
117-118i
146-147j
84-85
3m
3n
3o
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
152-153k
58-59
149-150l
135
C,m
H
C, H
C, H
C, H
C, H, N
C, H, N
C, H
C, H
C, H
96-97
143-144
20H15NO3S 132-133
20H15NO3S 160-161
The better in vitro activity in some assays observed
with compounds 4 having the phenyl ring directly
attached to the sulfur linkage compared to those with
methylene spacers may thus be directly related to the
improved stability also seen with those compounds.
20H14O4S
20H14O4S
21H16O3S
22H18O4S
22H18O3S
23H20O3S
151-152
189-190
155n
105-106
115-116
72-73
C, H
C, H
C, H
Su m m a r y a n d Con clu sion s
a
All new compounds displayed 1H NMR, FTIR, UV, and MS
spectra consistent with the assigned structure. b Elemental analy-
ses were within (0.4% of calculated values. c Literature58 mp 151-
10-Thio-substituted anthracenones were synthesized
and evaluated in various assays. Proper selection of the
10-thiophenyl substituent seemes to be critical for
potent in vitro activity, since this is strongly dependent
of the nature and position of the substituent on the
phenyl ring. 2-Aminophenyl and 4-hydroxyphenyl groups
are found to be optimal. Overall, compound 4h of this
series has the best biological profile both with respect
to inflammatory and hyperproliferative features of the
disease state and compares favorably in the cellular
assays with the antipsoriatic drug anthralin. The
amount of hydroxyl-radical formation by 4h is compa-
rable to that of anthralin, but contrary to this drug,7,10
4h does not enhance lipid peroxidation in model mem-
branes and is not cytotoxic as documented by the LDH
activity released from cytoplasm. It is one of the most
potent inhibitors of 12(S)-HETE formation in mouse
epidermis of the anthracenone class obtained to date
and also inhibits leukotriene biosynthesis in intact
PMNL. Furthermore, this compound is highly potent
and even more efficient as an inhibitor of human
keratinocytes growth and also more stable than anthra-
lin.
d
152 °C. Literature58 mp 120-122 °C. e Literature58 mp 152-153
g
°C. f C: calcd, 61.82; found, 61.07. Literature58 mp 92-94 °C.
h
j
i
Literature18 mp 176-180 °C. Literature58 mp 117-118 °C.
k
Literature58 mp 146-147 °C. Literature58 mp 154-155 °C.
l
m
n
Literature18 mp 145 °C. C: calcd, 71.84; found, 71.30. Lit-
erature18 mp 153 °C.
recorded on a MacLab data acquisition system (WissTech,
Germany) and analyzed with the software Peaks on an Apple
Macintosh computer.
Gen er a l P r oced u r e for th e Syn th esis of 10-(Alk ylth io)-
an d 10-[(ω-Su bstitu ted-P h en yl)th io]-1,8-dih ydr oxy-9(10H)-
a n th r a cen on es. 1,8-Dihydroxy-10-[(4-hydroxyphenyl)thio]-
9(10H)-anthracenone (4h). To a solution of 10-bromo-1,8-
dihydroxy-9(10H)-anthracenone16 (5, 305 mg, 1 mmol) and 0.1
mL of trifluoroacetic acid in dry CH2Cl2 (20 mL) was added
dropwise a solution of 4-mercaptophenol (252 mg, 2 mmol) in
dry CH2Cl2 (10 mL) under N2. The reaction mixture was
stirred at room temperature for 6 h. The solvent was removed
and the residue purified by column chromatography on silica
gel (E. Merck, 70-230 mesh) to provide yellow crystals (Table
3): 1H NMR (250 MHz, CDCl3) δ 11.83 (s, 2H), 7.49 (t, 2H, J
) 8.0 Hz), 7.01 (dd, 2H, J ) 7.5 Hz), 6.87 (dd, 2H, J ) 8.4
Hz), 6.55 (s, 4H), 5.35 (s, 1H), 4.91 (s, 1H); FTIR 3436 (OH),
1630 cm-1 (CO); MS m/ z 350 (11), 225 (100). Anal. C20H14O4S
(C, H).
log P Deter m in a tion . A standard reversed-phase HPLC
procedure was used. MeOH/water/HOAc (77/23/0.1), adjusted
to pH 5.5 with concentrated NH3, was used as eluant.
Calibration was performed as described.11 log P values as a
measure of lipophilicity are given in Table 3.
Deter m in a tion of th e Red u cin g Activity a ga in st 2,2-
Dip h en yl-1-p icr ylh yd r a zyl.11 To 1 mL of the test compound
solution (10-4 M) was added 1 mL of DPPH solution (10-4 M),
each in acetone/PBS (1/1 v/v), and the reduction of DPPH was
followed spectrophotometrically at 516 nm. Plots of the
reciprocal of DPPH concentrations against time gave straight
lines, and the second-order rate constants were obtained from
the slopes and are expressed as mean values (N ) 3-6).
Degr a d a tion of 2-Deoxy-D-r ibose. The deoxyribose assay
was conducted as described.11 The reaction mixtures contained
the following reagents at the final concentrations stated: 0.3
mL of KH2PO4-KOH buffer, pH 7.4 (30 mM), 0.2 mL of H2O
In conclusion, this study presents novel dual-function
inhibitors of both the 5- and 12-LO pathway and the
growth of human keratinocytes and suggests that
nontoxic agents of this type may be useful for the
treatment of psoriasis.
Exp er im en ta l Section
Melting points were determined with a Bu¨chi 510 melting
point apparatus and are uncorrected. 1H NMR spectra were
recorded with a Bruker Spectrospin WM 250 spectrometer (250
MHz), using tetramethylsilane as an internal standard. Fou-
rier-transform IR spectra (KBr) were recorded on a Nicolet
510M FTIR spectrometer. UV spectra were recorded on a
Kontron 810 spectrometer. Mass spectra 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

Antipsoriatic Anthrones with Modulated Redox Properties
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 16 3137
(3) Farber, E. M.; Nall, L. Psoriasis. A review of recent advances in
treatment. Drugs 1984, 28, 324-346.
(double distilled), 0.2 mL of 2-deoxy-D-ribose (2 mM), 0.2 mL
of FeCl3‚6H2O (0.1 mM), and 0.1 mL of anthracenone deriva-
tive (75 µM). After incubation for 2 h at 37 °C in a shaking
water bath, TBA-reactive material was measured at 532 nm.
Assa y of Lip id P er oxid a tion . Inhibition of lipid peroxi-
dation in bovine brain phospholipid liposomes was performed
essentially as described.1 The following reagents were added
at the final concentrations stated: 0.3 mL of KH2PO4-KOH
buffer, pH 7.4 (30 mM), 0.39 mL of H2O (double distilled), 0.2
mL of liposomes (1 mg/mL), 0.1 mL of AAPH (10 mM), and
0.01 mL of anthracenone derivative (variable concentrations).
After incubation for 1 h at 37 °C, TBA-reactive material was
measured at 532 nm.
Bovin e P MNL 5-Lip oxygen a se Assa y. Inhibition of 5-LO
was determined using Ca-ionophore-stimulated bovine PMNL
(107 cells/mL) as described.11 Test compounds were preincu-
bated for 15 min at 37 °C, and the concentrations of LTB4 and
5-HETE released after 10 min were measured by reversed-
phase HPLC analysis.
(4) Wiegrebe, W.; Mu¨ller, K. Treatment of psoriasis with anthroness
chemical principles, biochemical aspects, and approaches to the
design of novel derivatives. Skin Pharmacol. 1995, 8, 1-24.
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formation, challenges and prospects. Gen. Pharmacol. 1996, 27,
in press.
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Biochem. Pharmacol. 1988, 37, 4277-4280.
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and model membranes induced by anthralin (dithranol). Bio-
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species by dithranol, III. Dithranol, active oxygen species and
lipid peroxidation in vivo. Arch. Pharm. (Weinheim, Ger.) 1987,
320, 59-66.
Ep id er m a l 12-Lip oxygen a se Assa y. Preparation of the
epidermal homogenate and the 12-LO assay were performed
as described.22 Test compounds were preincubated for 5 min
at 37 °C, and the concentration of 12(S)-HETE formed after
10 min was measured by reversed-phase HPLC analysis. 12-
(S)-HETE was analyzed by chiral phase chromatography as
described.22
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anthrones with modulated redox properties. 1. Novel 10-
substituted 1,8-dihydroxy-9(10H)-anthracenones as inhibitors of
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Cell Cu lt u r e a n d Det er m in a t ion of Cell Gr ow t h .
HaCaT cells56 were cultivated, and the cell proliferation assay
was performed as previously described.1 After 48 h of incuba-
tion, 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 ) 3) with the control (N ) 6-8) activity: (1 - test
compound/control) × 100. Inhibition was statistically signifi-
cant compared to that of the control (Student’s t-test; P < 0.05).
Each IC50 value was derived by interpolation of a log inhibitor
concentration versus response plot using four or more concen-
trations of the compound, spanning the 50% inhibition point.
La cta te Deh yd r ogen a se Relea se.23 HaCaT cells were
incubated with the test compounds (2 µM) for 4 h at 37 °C.
Extracellular 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 ) 3, SD < 10%).
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Eluant: methanol/water/acetic acid (77/23/0.1, adjusted to pH
5.5 with NH3); flow rate 1 mL/min; 200 bar; detected at 254
nm.
Deter m in a tion of th e p Ka Va lu es.57 Absorption spectra
of the compounds were obtained in buffer solutions ranging
from pH 2.5-12.0. The pH values of the solutions were
measured with a Knick type 647 digital pH meter and an Ag/
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absorbance at pH 2.5 served as the absorbance of the neutral
species, whereas that at pH 12.0 as the absorbance of the
completely deprotonated species. The pKa values were deter-
mined from titration curves from absorbances at appropriate
wavelengths.
Ack n ow led gm en t. H.-S.H. thanks the National
Defense Medical Center, Taipei, Taiwan, R.O.C., for
financial support. We also thank I. Gawlik and K.
Ziereis for their kind help during the establishment of
the 12-LO and the keratinocyte test.
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